Peptide compositions for the treatment of HIV infection

ABSTRACT

The present invention provides for peptide conjugate compositions, methods of using the peptide conjugate compositions, and pharmaceutical compositions comprising the peptide conjugate compositions. The peptide conjugate compositions comprise peptides with amino acid sequences similar to the gp120 principal neutralizing domain (PND) of HIV, gp41, and Nef (p27) of HIV and carriers which enhance immunogenicity. The peptide conjugate compositions of the present invention may comprise a multivalent cocktail of several different peptide conjugates. Also provided by present invention is a method for reducing the level of HIV titers in a mammal by administering to the mammal a peptide composition of the present invention in an amount effective to reduce the level of HIV titers. The peptide conjugate compositions of the present invention induce prolonged antibody response in serum, a high level of antibody in the mucosa, and the production of cytotoxic lymphocytes. The peptide conjugate compositions of the present invention also elicit neutralizing antibodies and decrease viral loads in a subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.08/946,525, filed Oct. 7, 1997, now U.S. Pat. No. 6,139,843, which iscontinuation-in-part of U.S. patent application Ser. No. 08/785,696filed on Jan. 17, 1997, now abandoned, which is a continuation of U.S.patent application Ser. No. 08/655,376, filed May 30, 1996, nowabandoned, which is a continuation of U.S. patent application Ser. No.08/200,744, filed Feb. 23, 1994, now abandoned, which is acontinuation-in-part of U.S. patent application Ser. No. 07/837,781filed Feb. 14, 1992, now abandoned, which is a continuation-in-part ofU.S. patent application Ser. No. 07/681,624 filed Apr. 2, 1991, nowabandoned, the contents of each of which are hereby incorporated byreference in their entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under NIH Grant No. P30AI27741. As such, the government has certain rights in this invention.

BACKGROUND OF THE INVENTION

There have been recent advances in the use of retrovirus-derivedvaccines for the treatment of HIV. Specifically, a formalin-inactivatedwhole HIV vaccine has been developed which has conferred protection inMacaques. Immunization with vaccines potentiated with albumin hasresulted in the protection from clinical disease in eight out of ninemonkeys challenged with infectious, doses of HIV. Notably, protectioncould be achieved even in cases where entry of viruses is not prevented,suggesting that it may not be necessary to completely block infection inorder to have a successful vaccine.

Whole killed HIV vaccines have also been beneficial in the treatment ofchimpanzees who were previously infected by HIV. These chimpanzeesappear to have cleared the HIV infection in their blood streamsfollowing the vaccinations. Post-exposure immunization in humans hasalso been studied. These tests suggest that immunization may be used toprotect humans from HIV infections, and also to treat humans who havealready been infected with the virus. However, whole virus vaccines maycontain infectious particles. As a result, it may be safer to useessential components of the virus to confer protection. Epitopes of thevirus are one example of a safer, essential component of the virus. Morerecent studies have confirmed that partial protection from infection canbe achieved also by gp120 and gp160 derived vaccines. (Desrosiers, R. C.et al. Proc. Nat'l Acad. Sci. USA, 86:6353 (1989), Kestler, et al.Science, 248:1109 (1990); Murphey-Corb, M. Science, 246:1293 (1989)).

It has long been recognized that peptide epitopes of amino acidsconjugated to immunogenic carriers can elicit high levels of highaffinity antipeptide antibodies. (See Talwar, G. P., Bloom, B. et al.,“Biological and Clinical Aspects of Reproduction”, Exceptor Med. Series394:2224-2232 (1987), in which the beta chain of human chorionicgonadotropin was conjugated to tetanus toxoid to produce anantifertility vaccine.)

The carriers to which peptides are conjugated in this invention have allbeen used as immunogenic carriers in animals, and some have been used inhumans. By way of example, the purified protein derivative (PPD) oftuberculin from Mycobacterium tuberculosis, which is the preferredcarrier of the invention, is a unique immunologic reagent, becausevirtually everyone in the world with a functional immune response whohas been exposed to BCG or M. tuberculosis infections will have a T-cellmediated, delayed-type hypersensitivity response to minute amounts ofPPD. Tuberculin-PPD conjugates have been utilized in the past. Micepre-sensitized or “primed” with BCG can produce high levels ofantibodies to peptide or carbohydrate epitopes conjugated to PPD. Ofparticular interest are studies on the NANP repeating epitope of the P.falciparum circumsporozoite antigen, which is immunogenic in only twostrains of mice. Conjugating the NANP repeating peptide to PPD elicitsthe production of antibody titers greater than 1:1000 in geneticallynon-responder strains to the NANP epitope. This degree of response iscomparable to that seen in responder strains given the peptide conjugatein complete Freund's adjuvant. (See Lussow et al., “Use of TuberculinPurified Protein Derivative-Asn-Ala-Asn-Pro Conjugate in BacillusCalmette-Guerin Primed Mice Overcomes H-2 Restriction of the AntibodyResponse and Avoids the Need for Adjuvants,” Proc. Nat'l Acad. Sci. USA87 (1990)).

Pseudomonas aeruginosa exotoxin A (toxin A) has been used effectively asa carrier in conjugate vaccines. Conjugates made with this carrier havehigher immunogenicity, especially when coupled with the recombinantprotein R32 to create an immune response against the sporozoite stage ofPlasmodium falciparum. Pseudomonas aeruginosa exotoxin A may be purifiedfrom the supernatant of fermentor-grown cultures of Pseudomonasaeruginosa PA 103. Toxin A has been classified as a superantigen basedupon results in animals. Toxin A can be completely and irreversiblydetoxified by covalent coupling to adipic acid dihydrazide (ADH), a 4carbon spacer molecule. This step destroys the ADPR-transferase activityof the toxin molecule, hence rendering it nontoxic. The non-reactedhydrazide group can be used to covalendy couple haptens to toxin A.

To date, the following haptens have been coupled to toxin A by theinventors by the use of ADH and carbodiimide as a coupling agent: (1)small molecular weight polysaccharides from P. aeruginosa andEscherichia coli; (2) the immunodominant (NANP)³ repeat from Plasmodiumfalciparum circumsporozoite; and (3) a recombinant protein, termedR32LR, which contains multiple NANP and NVDP repeats from P. falciparum.

Approximately 5,000 subjects have been immunized by the inventors withtoxin A-containing vaccines produced by the inventors. As much as 400 mgof toxin A have been administered per dose, with multiple (3) dosesgiven to subjects. These vaccines have been very well tolerated. Mild tomoderate, transient local reaction occur in 0.25% of vaccines. Systemicreactions occur in 0.1-2%. Abnormal blood chemistries have not beenassociated with these vaccines.

Keyhole Limpet Hemocyanin (KLH) is a high molecular weight protein whichis purified from megathura crenulata. KLH has many available primaryamines from lysine residues which facilitate protein conjugation. KLH ishighly immunogenic, and because of its availability of primary amines,is ideal for protein conjugation.

Tetanus and diphtheria toxoids have also been used successfully asprotein carriers. Diphtheria toxoid has been used with a synthetic 31amino acid N-terminal peptide. Both tetanus toxoid and diphtheria toxoidhave proved to be effective carriers in humans for the poorlyimmunogenic carbohydrate antigen of Hemophilus influenza b.

The recombinant core antigen of hepatitis B has the capability ofself-assembling into 27 millimeter particles which are highlyimmunogenic in experimental animals. These HBV core particles may beconjugated directly with peptides, using recombinant DNA technology.Fusion proteins can be produced between the HBV core antigen and definedsequence peptides with high epitope density, which lead to high titerantibodies, as well as to long lasting neutralizing antiviral immunity.Hepatitis B core antigens and self-assembled HBc-HIV peptide fusionprotein may be used as protein carriers.

It has been established that the major antigenic component of themycobacterial cell wall is a protein which consists of a polypeptidemonomer of between 10 and 16 Kd, the amino terminal sequence of whichreveals that it is related to the GroES heat-shock protein present inmany bacteria. It has been indicated that the major antigenic componentof mycobacteria recognized by CD4+ T-cells is associated with the cellwall. BCG cell wall (purified) may be used as a protein carrier. BCG mayalso be used to prime animals or humans prior to vaccination. BCGpriming enhances the humoral and cellular responses induced byvaccination.

Currently, the only adjuvant licensed for use in man is alumina. In thepresent invention, alumina may be used with the conjugates which includethe carriers Pseudomonas aeruginosa exotoxin A, KLH, and tetanus anddiphtheria toxoids. Aluminum-based gels such as Al(OH)₃ and AlPO₄, aswell as liposomes may be used as adjuvants with the carrier Pseudomonasaeruginosa exotoxin A in the present invention. PPD conjugates can begiven in saline with no further adjuvants to tuberculin-positivesubjects. For the BCG cell wall and HBc antigen conjugates, it is likelythat adjuvants would be required. Ribi adjuvant containing trehalosedimycolate and 2% squalene may be used as an adjuvant. Alternatively, ifstudies on the long-term safety of incomplete Freund's adjuvant orISCOM's adjuvant indicate their safety and efficacy in humans, theseadjuvants may be used. Microencapsulation technology usingpolyactide/polyglycolide biodegradable polymers may also be used as anadjuvant.

Because a significant amount of HIV-1 transmission occurs from cell tocell (see McCune, “HIV-1: The Infective Process in vivo”, Cell, Vol. 64,pp. 351-363 (1991)), neutralizing antibodies alone cannot preventclinical infection. Furthermore, since the majority of HIV-1transmission occurs via the mucosal route, effective mucosal immunity isnecessary for protection from HIV-1 infection. The effectiveness of anHIV-1 vaccine depends upon its capacity to induce HIV-1 specific cellmediated immunity and humoral immunity in both serum and in the mucosa.

To date, no preventive vaccine has been reported which induces humoral,cellular and mucosal immune response. Hence, it is desirable to developa vaccine which induces antibody (serum and mucosal) as well asPND-specific T cell (CTL) response after immunization therewith.

Current results with post-infection HIV-1 recombinant gp120 and gp160(Salk J, et al. Science 1993; 260: 1270-72; Redfield R R, et al. N EnglJ Med 1991; 324: 1667-84; Valentine F T, et al. J Infect Dis 1996; 173:1336-46; Eron J J, et al. Lancet. 1996; 348: 1547-51; Haynes B F Lancet.1996; 348:933-37; Haynes B F Lancet 1996; 348: 1531-2) vaccines arediscouraging. (Redfield R R, et al. N Engl J Med 1991; 324: 1667-84;Valentine F T, et al. J Infect Dis 1996; 173: 1336-46; Eron J J, et al.Lancet 1996; 348: 1547-51). In light of the enormous turnover of HIV-1virions and CD4 cells (Saag M S, et al. Nat Med 1996; 625-29; Mellors JW, et al. Ann Intern Med 1995; 122: 573-79; Ho Dd, et al. Nature 1995;373: 123-126; Fauci A S Nature 1996; 384: 529-33) it was suggested thatthe prospect of ever inducing a more effective anti-HIV-1 immunity wasslim in an immune system that is already working overtime. (Haynes B FLancet 1996; 348:933-37; Haynes B F Lancet 1996; 348: 1531-2; Haynes BF, et al. Science 1996; 271: 324-8).

In contrast to this bleak outlook are observations of immune responsesin long term non-progressors that are absent or decreased in rapidprogressors (Haynes B F Lancet 1996; 348:933-37; Haynes B F Lancet 1996;348: 1531-2; Haynes B F, Science 1996; 271: 324-8) and recent studies ininfants have indicated the existence of abortive infections. (Bryson YJ, N Engi J Med 1995; 332: 833-8; Roques P A, AIDS 1995; 9: F19-26.) Thedevelopment of our vaccine was based on studies showing a correlationbetween high affinity antibodies to gp120 or to the V₃ loop and reducedmaternofetal HIV-1 transmission. (Goedert J J, Lancet 1989; ii: 1351-53;Rubinstein A, et al. AIDS 1995; 9: 243-51). The V₃ loop is known toparticipate in vital viral properties such as cell tropism and cellfusion. Antibodies to the V₃ loop decline with disease progression,while antibodies to whole gp160 remain stable. (Fenouillet E, Clin ExpImmunol 1995; 99: 419-24). Guinea pig immune sera against a BCG vectorsecreting the V₃-Primary Neutralizing. Domain (PND) blocked HIV-1infection in SCID/hu mice (Honda M, et al. Proc Natl Acad Sci USA 1995;92: 10693-97) and a monoclonal antibody to the V₃ loop protected chimpsagainst HIV-1 infection. (Emini E A, et al. Nature (London) 1992; 355:728-3018).

Thus, there remains a need for the discovery and development of peptidecarrier conjugate vaccines capable of inducing prolonged antibody immuneresponse. There is an additional need for vaccines which are capable ofinducing a serum humoral immune response, mucosal humoral immuneresponse and the production of cytotoxic lymphocytes. In addition, thereis a great need for the discovery and development of an effective methodof treatment and prevention of HIV infection which will reduce the viralload in a subject, thereby preventing or limiting the progression of thedisease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the immunoreactivity of mice vaccinated withconjugates of KLH and five different peptides: 282 (GPGRAFGPGRAFGPGRAFC)(SEQ ID NO:5), 283 (IYIGPGRAC) (SEQ ID NO:2), 284 (IAIGPGRAC) (SEQ IDNO:3), 285 (IHIGPGRAC) (SEQ ID NO:4) and MN (KRIHIGPGRAFYT) (SEQ IDNO:1).

FIG. 2 represents the affinity/avidity of mice with the highest titersof antibody to conjugates of KLH and peptides 282 (GPGRAFGPGRAFGPGRAFC)(SEQ ID NO:5), 283 (IYIGPGRAC) (SEQ ID NO:2), 284 (IAIGPGRAC) (SEQ IDNO:3) and 285 (IHIGPGRAC) (SEQ ID NO:4).

FIGS. 3A-3C represent the results of competition between peptides 282(GPGRAFGPGRAFGPGRAFC) (SEQ ID NO:5) and MN (KRIHIGPGRAFYT) (SEQ ID NO:1)with peptide 283 (IYIGPGRAC) (SEQ ID NO:2) in mouse 283-5 (FIG. 3A).Peptide 282 competed effectively, and peptide MN did not. FIGS. 3B and3C also represents the results of competition between peptides 282 (FIG.3B) and MN with peptide 284 (IAIGPGRAC) (SEQ ID NO:3) (FIG. 3C) in mouse284—4. Both peptides 282 and MN competed effectively.

FIGS. 4A and 4B represent reciprocal antibody titers, measured by PNDELISA (FIG. 4A) and affinity of antibodies as measured by theantigen-limited ELISA (FIG. 4B). Reactivities with PND coating of theELISA wells with 100 mg or less were considered high affinity/highavidity.

FIG. 5 represents the cross-reactivity of serum antibodies from vaccinesubject #17 with MN-PND, with heterologous PND peptides and with theGPGRAF motif. Specific absorbance was measured in ELISA plates in whichwells were coated with the respective peptide. The open bars representthe control, and the shaded bars represent the subject.

FIG. 6 represents the neutralization of HIV-1_(MN) by serum fromPPD-MN-PND immunized subjects as compared to normal human serum. Serawere diluted at 1:10 and 1:100 before incubation with HIV-1_(MN). P24levels were measured in supernatants of cultured HIV-1_(MN) infected H9cells.

FIG. 7 represents proliferative responses to MN-PND. 10⁵ peripheralblood lymphocytes of vaccines were stimulated with 200 mg of PND for 7days. Cellular proliferation was assessed by determining theincorporation of [³H] thymidine added during the last 16 hours ofculture.

FIG. 8 represents CTL response as shown by percent of net specificlysis.

FIGS. 9A-9E represent the results of immunization of rabbits withvarious peptides coupled to KLH. FIG. 9A shows the results using theKRIHIGPGRAFYT (SEQ ID NO:1) peptide; FIG. 9B the GPGRAFGPGRAFGPGRAFCpeptide (SEQ ID NO:5); FIG. 9C the IYIGPGRAC (SEQ ID NO:3) peptide; FIG.9D the IHIGPGRAC (SEQ ID NO:4) peptide; and FIG. 9E the IAIGPGRAC (SEQID NO:3) peptide.

FIGS. 10A-10E represent the results of immunization of rabbits withvarious peptides coupled to KLH. FIG. 10A represents the results usingthe KRIHIGPGRAFYT (SEQ ID NO:1) plate; FIG. 10B the GPGRAFGPGRAFGPGRAFC(SEQ ID NO:5) plate; FIG. 10C the IYIGPGRAC (SEQ ID NO:2) plate; FIG.10D the IHIGPGRAC (SEQ ID NO:4) plate; and FIG. 10E the IAIGPGRAC (SEQID NO:3) plate.

FIG. 11 represents the antibody results of immunization of BCG-primedand non BCG-primed guinea pigs with MN-PND coupled to PPD or toxin A.

FIGS. 12A-12G set forth antibody responses following three monthlyimmunizations with the PPD-pentapeptide-PND vaccine (OD=optical densityat 490/650 nm). ELISA plates were coated with the respective peptide atpeptide concentration of 1 μg/ml per well. FIG. 12A represents theresults of immunization of patient #1; FIG. 12B of patient #2; FIG. 12Cof patient #3, FIG. 12D of patient #4; FIG. 12E of patient #5; FIG. 12Fof patient #6; and FIG. 12G of patient #7.

FIGS. 13A and 13B indicate the induced antibody affinity for the MNpeptide in patient #2 (FIG. 13A) and patient #5 (FIG. 13B) before andfollowing immunizations with the PPD-pentapeptide-PND vaccine.Microtiter ELISA plate wells were coated with decreasing amounts of theMN peptide, from 10,000 ng/ml to 10 ng/ml. An OD over background at ≦100ng/ml was considered to indicate high affinity.

FIGS. 14A-14G indicate neutralization of a Clade B primary isolate(HIV-1-59) by vaccinees' sera, pre and post immunization. The shift ofthe curves to the left and towards the horizontal axis with time postimmunizations indicates a temporal increase in virus neutralization.FIG. 14A represents the results of immunization of patient #1; FIG. 14Bof patient #2; FIG. 14C of patient #3, FIG. 14D of patient #4; FIG. 14Eof patient #5; FIG. 14F of patient #6; and FIG. 14G of patient #7.

FIGS. 15A-15G set forth the effect of PPD-pentapeptide-PND immunizationon viral load (NASBA assay) RNA copies/ml. FIG. 15A represents theresults of immunization of patient #1; FIG. 15B of patient #2; FIG. 15Cof patient #3, FIG. 15D of patient #4; FIG. 15E of patient #5; FIG. 15Fof patient #6; and FIG. 15G of patient #7.

FIGS. 16A and 16B set forth the immunoreactivity pattern of serum froman HIV(+) PPD (+) patient before (FIG. 16A) and after (FIG. 16B) 2intradermal immunizations with hexa-PND-peptide-PPD. Antigen limitedELISA utilizing PND (p282-p357) peptides of the vaccine. Note theincrease of OD to 3 of the PND-peptides.

FIG. 17 sets forth the neutralization of HIV-1 primary isolates: in PBMCby serum from HIV(+) volunteers immunized with the PPD-hexa-PND-AIDSvaccine. The shift of the curves to the left indicates neutralizationincrement post: vaccination. Using the ACTG consensus log reductionassay, the TCID₅₀ reduction was calculated according to theSpearman-Kraber method, yielding in this volunteer a 267 fold increasein serum neutralizing capacity at 12 months post vaccinations.

SUMMARY OF THE INVENTION

The present invention provides for peptide conjugate compositions whichcomprise peptides with amino acid sequences similar to the gp120principal neutralizing domain (PND) of HIV, gp41, and Nef (p27) of HIVand carriers which enhance immunogenicity, such conjugates to be used incompositions for the treatment and prevention of HIV infection. Thepeptide conjugate compositions of the present invention may compriseeither a single peptide conjugate alone or a cocktail of severaldifferent peptide conjugates.

The present invention further provides a method for reducing the levelof HIV titers in a mammal by administering to the mammal a peptidecomposition of the present invention in an amount effective to reducethe level of HIV titers. The peptide conjugate compositions of thepresent invention induce prolonged antibody response in serum, a highlevel of antibody in the mucosa, and the production of cytotoxiclymphocytes. The peptide conjugate compositions of the present inventionalso elicit neutralizing antibodies and decrease viral loads in asubject.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for peptide conjugate compositions to beused in the treatment and transmission prevention of HIV. The peptideconjugate compositions of the present invention are made by conjugatingpeptides with amino acid sequences similar to specific regions of HIVand carriers so as to form peptide conjugates.

The peptide conjugate compositions of the present invention not onlyinduce the production of high affinity/avidity antibodies in the serum,but induce the production of such antibodies for extensive periods oftime, and also induce the production of antibodies in the mucose. Thepeptide conjugate compositions of the present invention also induce cellmediated immune response by inducing the production and proliferation ofcytotoxic lymphocytes. Further, the peptide conjugate compositions ofthe present invention induce in subjects an increase or emergence ofneutralizing antibodies to primary HIV isolates and autologous HIVisolates, and reduce HIV viral load in these subjects.

The present invention provides a method for reducing the level of HIVtiters in a mammal by administering to the mammal a peptide compositionin an amount effective to reduce the level of HIV titers. The peptidecomposition is administered to the subject prior to, or subsequent to,HIV infection. The peptide composition comprises at least one peptidecoupled to an immunogenic carrier, wherein said peptide is selected fromthe group consisting of KRIHIGPGRAFYT (SEQ ID NO:1), RSIHIGPGRAFYA (SEQID NO:6), KSITKGPGRVIYA (SEQ ID NO:7), KGIAIGPGRTLYA (SEQ ID NO:8),SRVTLGPGRVWYT (SEQ ID NO:9), and HIV strain variants thereof. Thepeptide composition may comprise at least two of the above peptidescoupled to an immunogenic carrier. In a preferred embodiment of theinvention, the peptide composition comprises at least five of the abovepeptides coupled to an immunogenic carrier. The peptide composition mayfurther comprise at least one peptide selected from the group consistingof LLELDKWA (SEQ ID NO:10), RPMTYK (SEQ ID NO:11), GGKWSK (SEQ IDNO:12), PGPGIRY (SEQ ID NO:13), GPGIGPGV (SEQ ID NO: 14), and HIV strainvariants thereof.

Also provide by the present invention are pharmaceutical compositioncomprising a peptide composition comprising at least one of thefollowing peptides coupled to an immunogenic carrier: KRIHIGPGRAFYT (SEQID NO:1), RSIHIGPGRAFYA (SEQ ID NO:6), KSITKGPGRVIYA (SEQ ID NO:7),KGIAIGPGRTLYA (SEQ ID NO:8), SRVTLGPGRVWYT (SEQ ID NO:9), LLELDKWA (SEQID NO:10), RPMTYK (SEQ ID NO:11), GGKWSK (SEQ ID NO:12), PGPGIRY (SEQ IDNO:13), GPGIGPGV (SEQ ID NO: 14), and HIV strain variants thereof. Thepharmaceutical composition may fturther comprise at least one peptideselected from the group consisting of LLELDKWA (SEQ ID NO:10), RPMTYK(SEQ ID NO:11), GGKWSK (SEQ ID NO:12), PGPGIRY (SEQ ID NO:13), GPGIGPGV(SEQ ID NO: 14), and HIV strain variants thereof. The peptidecomposition preferably is present in the pharmaceutical composition inan amount effective to reduce HIV titers in a mammal that thepharmaceutical composition is administered to.

HIV strain variants of the peptides of the present invention are hereindefined as peptides corresponding to the peptides of the presentinvention which are obtained from other strains of HIV. These peptidesmay vary from the peptides of the present invention by 1-3 amino acidsat either or both the carboxy and amino terminal ends of the peptide.

Carriers which may be used for conjugation with the peptides of thisinvention include purified protein derivative (PPD) of tuberculin fromM. tuberculosis, Pseudomonas aeruginosa exotoxin A (toxin A), keyholelimpet hemocyanin (KLH), filamentous hemagglutinin (FHA) of bordetellapertussis, tetanus toxoid, diphtheria toxoid, hepatitis B core antigen,T helper cell (Th) epitopes of tetanus toxoid (TT) and BacillusCalmette-Guerin (BCG) cell wall. The preferred carriers of thisinvention are PPD and toxin A. In addition, an adjuvant, such asaluimina, Ribi, Freund's, Iscom's or microencapsulation technologyadjuvant may be used with these carriers.

Other carriers which may be used for conjugation with peptides of thisinvention are the recombinant 10 kDa, 19 kDa and 30-32 kDa proteins fromM. leprae and from M. tuberculosis, or any combination of theseproteins. The 10 kDa, 19 kDa and 30-32 kDa proteins are antigensrecognized by most M. tuberculosis and M. leprae-reactive human T celllines and cell wall reactive cell clones. The 10 kDa protein is 44%homologous to the heat-shock protein (HSP-Groes) of E. coli. The 19 kDaprotein has a lipidation signal in it, and was shown to stimulate IL-2production (Dr. Barry Bloom, unpublished data).

The carriers may be coupled to the peptides of the present inventionusing techniques know to one skilled in the art. In a preferredembodiment of the invention, PPD is coupled to a peptide of the presentinvention using glutaraldehyde. Using this method allows for randomconformational and linear presentations of the peptides and cantherefore be recognized by a broader range of MHC-I and MHC-IImolecules. The coupling method also allows for intracellular processingof the peptide in the cytosol to promote MHC-I responses. In a preferredembodiment of the invention, each peptide in a multiple, multivalentpeptide mixture is coupled separately to PPD and a mixture of all thecoupled peptides is then prepared. This method of conjugation alsoincreases the ratio of peptide to PPD and reduces the delayed typehypersensitivity skin response (DTHR) to PPD, allowing for the use ofmuch higher PPD doses intradermally without local adverse effects. In apreferred embodiment of the invention, 2-5TU are used for skin testingwith PPD, and 12-25TU is used for the vaccination and is injected intoone site.

Proliferative responses of PBNC to the 10 kDa antigen from M.tuberculosis are similar to those induced by whole M. tuberculosis, andgreater than those elicited by other proteins from M. tuberculosisculture filtrates. 10 kDa antigens are identical to those defined forBCG-a. The 10 kDa antigen elicits IFN production by PBMC of healthytuberculin reactors and by plural fluid mononuclear cells. In addition,T cell clones reactive to the 10 kDa protein from M. leprae alsorecognize epitopes that are cross-reactive with the antigen of M.tuberculosis. Further, the 10 kDa protein elicits strong delayed-typehypersensitivity reactions (DTHR) in guinea pigs sensitized to M. lepraeand to BCG.

Purified 32 kDa protein from filtrates of M. tuberculosis and BCG isalso believed important for protective immunity. T cell derivedlymphokines, such as interferon, play an essential role in the controlof disease. It has been shown that all tuberculoid leprosy patients withbenign disease course and controls have a marked parallelism ofresponsiveness towards whole M. leprae and purified antigen 85, which isthe major secreted 30-32 kDa protein antigen from M. bovis BCG. Thisresponsiveness is comprised of T cell proliferation and IFN production.The inventors have discovered that BTHR to the carrier PPD is strongerthan that to 10 kDa M. tuberculosis and 10 kDa M. leprae proteins.However, the lymphoproliferative response to IFN secretion in sensitizedanimals and subjects appear to be at least identical between crude PPDand clone- expressed gene products of M. tuberculosis. Hence, thecoupling of 10 kDa to the peptides of this invention may allow forimproved protective in vivo responses to HIV as well as reducedunpleasant DTHR skin reactions in vaccines.

The inventors immunized BCG-primed guinea pigs at monthly intervals with10 kDa M. leprae- and 10 kDa M. tuberculosis-PND conjugates. Thevaccines were given at doses of 25 mg and 125 mg, respectively. Theguinea pigs demonstrated a delayed type skin reactivity to the peptides.In addition, there was an in vitro lymphocyte antigenic response to thepeptides with which the animals were immunized. Hence, a cell-mediatedimmune response may be acquired using 10 kDa M. leprae and 10 kDa M.tuberculosis proteins conjugated to PND.

In order to prepare the peptides of this invention, it is necessary todetermine which strains of HIV infect the subjects to be treated, andwhich peptides of each strain would most effectively induce theproduction of high affinity/avidity neutralizing and/or protectiveHIV-specific antibodies. There are many different strains of HIV, anddifferent strains are prevalent in different geographic areas.Therefore, the peptides in the vaccines of this invention may begeographically specific. For example, the MN strain of HIV has a highdegree of prevalence in the United States. Therefore, peptides from theMN strain of HIV are effective in conjugates used to vaccinate subjectsin the United States. These particular peptides and conjugates may notbe effective in different geographic areas where other HIV strains areprevalent, and may have to be adjusted according to the most prevalentstrains evolving in certain geographic areas. Another example is that inthe United States, high affinity/avidity neutralizing and/or protectiveHIV-specific antibodies against the gp120 PND of the MN strain of HIVhave been associated with protection. from materno-fetal HIVtransmission. Therefore, peptides from the MN strain of HIV may be usedin conjugates to prevent materno-fetal transmission of HIV in the UnitedStates.

Peptides which may be employed in the vaccines of this inventioninclude, for example, those obtained from the gp120 PND of HIV. Thepeptides selected may include amino acids 116-131, 307-319 and 470-490of gp120 of MN-HIV. The most effective sequences to induce theproduction of high affinity/avidity neutralizing and/or protectiveHIV-specific antibodies, which are the preferred peptide sequences forthe conjugates of this invention, are as follows:

SEQ. ID. NO. 1 KRIHIGPGRAFYT

SEQ. ID. NO. 2 IYIGPGRAC

SEQ. ID. NO. 3 IAIGPGRAC

SEQ. ID. NO. 4 IHIGPGRAC

SEQ. ID. NO. 5 GPGRAFGPGRAFGPGRAFC

SEQ. ID. NO. 6 RSIHIGPGRAFYA

SEQ. ID. NO. 7 KSITKGPGRVIYA

SEQ. ID. NO. 8 KGIAIGPGRTLYA

SEQ. ID. NO. 9 SRVTLGPGRVWYT

Other peptides which may be used in the vaccines of this invention maybe from the gp160 of HIV-1. Epitopes of HIV-1 gp160 have been shown tobe recognized by T cells of HIV-1 infected subjects. See Clerici et al.,“Interleukin-2 Production Used to Detect Antigenic Peptide RecognitionBy T-Helper Lymphocytes From A symptomatic HIV-SeropositiveIndividuals”, Nature, Vol. 339, pages 383-386 (1989). In addition,epitopes of HIV-1 gp160 are involved in affecting the course of HIV-1infection. Since the carrier PPD recruits T cell help, the coupling ofpeptides to PPD whose sequences correspond to an HIV-1 T cell epitopewould result in a vaccine that is a powerful inducer of T cell responseto the HIV-1 T cell epitope. Therefore, epitopes such as T1, describedby Clerici et al., and HIV-1 cytotoxic T lymphocyte (CTL) epitopespresent on reverse transcriptase may be used in the vaccines of thisinvention. (See Hosmalin et al., “An Epitope of HIV-1 ReverseTranscriptase Recognized by Both Mouse and Human CTL”, Proc. Natl. AcadSci, USA, Vol. 87, page 2344 (1990)). Further, these peptides, or otherpeptides with similar properties, may be coupled to PPD and includedwith the MN-PND-PPD cocktail vaccine.

Other peptides which may be used in the vaccines of the presentinvention include V3 loop peptides, which are linear peptides from aminoacids 307-319 associated with the V₃ loop region. These peptides may,for example, represent THAI-I, THAI-II, MN RF, NY-5, CDC-42 and ARV: 2.

Another peptide that may be used in the vaccines of the present.invention is a gp41 peptide, for example, the 6 amino acid peptideELDKWA shown by Katinger to be the target of a cross-neutralizingmonoclonal antibody may be used in a modified form. This peptidesequence by itself was found by many investigators to benon-immunogenic. The inventors have discovered that the addition to twoLL (LLEDKWA) (SEQ ID NO:10) in a repetitive motif of 16 amino acids issurprisingly highly immunogenic.

Other peptides which may be employed in the vaccines of the presentinvention are Nef peptides. The selection of Nef epitopes is based onthe conserved features of Nef sequences, on their functional properties(Nixon, D. F., et al. AIDS 5:1049, 1991; Cheingsong-Popov, R., et al.AIDS Res. & Human Retrov. 6:1099, 1990; Schneider, et al. AIDS Res. &Human Retrov. 1:37, 1991; Siakkou, H., et al. Arch. Virol 128:81, 1993;Culmann, B., et al. J. Immunol. 146:1560, 1991; Yu, G., et al. Virology187:46, 1991; Shugars, D. C., et al. J. Virol. 67:4639-4650, 1993;Venet, A., et al. AIDS Res. & Human Retrov. S41, 1993; Robertson, M. N.,et al. AIDS Res. & Hunman Retrov. 9:1217-23, 1993) and on sequencesfound missing in patients with “non-virulent” HIV-1 disease (long-termnon-progressors). Sequences known to induce both humoral and CTLresponses (Cheingsong-Popov, R., et al. AIDS Res. & Human Retrov.6:1099, 1990; Schneider, et al. AIDS Res. & Human Retrov. 1:37, 1991;Siakkou, H., et al. Arch. Virol 128:81, 1993; Culmann, B., et al. J.Immunol. 146:1560, 1991; Robertson, M. N., et al. AIDS Res. & HumanRetrov. 9:1217-23, 1993) are selected. Other peptides that may beemployed include, for example:

a. RPMTYK (SEQ ID NO: 11)—a highly conserved recognition site forphosphorylation by protein kinase C:

b. GGKWSK (SEQ ID NO:12) —a nearly invariant myristilation site whichlies on the external surface of the folded nef protein.

c. PGPGIRY (SEQ ID NO:13) and GPGIGPGV (SEQ ID NO: 14) located atpositions 13-138, a highly conserved region predictive of a beta turn(Shugars, D. C., et al. J. Virol. 67:4639-4650, 1993).

Because soluble peptides are poor immunogens due to their lack of T celland B-cell reactive epitopes and their low molecular weight, thepeptides of this invention are conjugated with different carriers toenhance immunogenicity. The methods by which these peptides areconjugated with the carriers include disulfide linkages through a Cterminal peptide cysteine linkage, coupling with glutaraldehyde solutionfor two hours, coupling with tyrosine, or coupling with water solublecarbodiimide.

Where the carrier is PPD, PPD-negative subjects should be primed withBCG prior to conjugate vaccination. In order to determine whether asubject is PPD-negative or PPD-positive, either proliferative responsetesting or skin testing may be performed. For proliferative responsetesting, if a subject exhibits proliferative responses in vitro toperipheral blood lymphocytes to PPD, that subject is PPD-responsive. Forskin testing, a subject should be exposed to intradermal 5 TU and, ifnegative, to 20 TU of PPD. If a subject is PPD-positive, there is noneed to prime with BCG. If a subject is considered PPD-negative, thatsubject should receive a BCG immunization four to six weeks prior toconjugate vaccination. After BCG immunization, PPD testing should againbe performed. If the results of the PPD test are positive, then theconjugate vaccine is administered to the subject.

If subjects are HIV-negative and PPD-negative, there are no limitationson BCG priming. However, if subjects are HIV-positive and PPD-negative,they may be primed with BCG only if they are still asymptotic and/ortheir CD4 T-cell counts exceed 200. Subjects who are HIV-positive,PPD-negative and exhibit advanced symptoms should not be primed withBCG. In addition, it is recommended that the standard British orJapanese BCG vaccines be used for priming, since these vaccines rarelyinduce adverse reactions in subjects.

The peptide-carrier conjugates of this invention may be administered invaccine form, suppository form, intradermally, subcutaneously, orally,or by any other suitable route. The vaccines may be administered in anysuitable form including liquid form, or in timed-release, pulse-releaseor slow-release mechanisms, such as virosomes. Virosomes encaseviral-specific antigens in their systems and then react strongly withmacrophages. Such virosomes may comprise 1-10 mg of PND peptide coupledto PPD or influenza hemagglutinin (HA), or PND in a free stateassociated with but not covalendy coupled with PPD or HA, 1-10 mg of HAisolated from a human strain of influenza virus, 0.1-1 mg neuraminidase(NA) isolated from a human strain of influenza virus, 0.25-0.75 mgkephalin and 100-140 mg lecithin.

The vaccines of this invention should be administered in dosageconcentrations and regimens which induce high affinity/avidity antibodyresponse. This approach is in contrast to accepted vaccination practiceswherein the general goal is to elicit a higher total antibody responsewithout specific regard to affinity/avidity of the antibody produced.Accepted vaccination practices are usually designed so as to incorporatea comparatively large amount of antigen and a dosing scheme to elicitmaximal antibody titers over the shortest period of time. In contrast,the dosage and regimen schedules of the present invention allow for alimiting amount of antigen and an extended vaccination schedule so as toselect for the induction of high affinity/avidity and/or neutralizingantibodies.

The vaccines of the present invention may be administered at a singlesite, or at multiple sites. Where the carrier to be employed in thevaccine is PPD, the preferred concentration of PPD is 2 IU-100 IU perhuman dose, and the preferred dosage range of the peptide is 0.1-2.5 mg.When skin testing is employed, 2-5 TU are used. The preferred ratio ofpeptide to PPD is in the range of 0.1:1-2:1. Where the carrier to beused in the vaccine is toxin A, the preferred dose range of peptide isin the range of 1-50 mg per human dose, and the preferred molar range ofpeptide to toxin A is in the range of 2:1-20:1.

Generally, small doses of vaccine antigen given at an appropriateschedule will selectively be taken up and hence stimulate immunelymphocytes expressing high affinity/avidity antibodies on their cell'ssurface. This is particularly true after a subject is primed with agiven antigen either by natural exposure or by prior vaccination.Following primary immunization, only a comparatively small subset ofprimed lymphocytes will have high affinity/avidity antibodies expressedon their cell's surfaces. By subsequently immunizing with a small doseof vaccine antigen as a booster, the limiting antigen willpreferentially be taken up by high affinity/avidity cells surfaceantibodies which will result in the expansion of such cells with theresult being a preferential induction of high affinity/avidityantibodies. Multiple closely-spaced doses, especially with a largeamount of antigen, can induce suppressor T cells which down-regulate theimmune response, especially to a booster dose of vaccine. To circumventthis problem, a series of primary immunizations (2-3 doses of vaccinesspaced 2-4 weeks apart) should be given to prime the immune system. Abooster dose of vaccine is not given until at least 3-4 months haveelapsed since the first dose of vaccine was given. In this manner, thepopulation of suppressor T cells rising via primary immunization willhave declined. Therefore, the preferred immunization schedule is a doseof vaccine given on days 0, 28 and 84. Subsequent booster doses can begiven at 84-160 day intervals to maintain immunity.

The vaccines of this invention may comprise either a single peptidecarrier conjugate or a cocktail of several different peptide carrierconjugates. In addition, it is possible to broaden neutralizationactivity by first immunizing with one peptide, for example MNPND(KRIHIGPGRAFYT) (SEQ ID NO:1) and then boosting at a later date with adifferent peptide conjugated to a carrier (for example, (GPGRAF)(SEQ IDNO:16) 3C conjugated to PPD). By doing this, a stronger antibodyresponse against the GPGRAF (SEQ ID NO:16) epitope is induced, and sincethere is cross-neutralization, a broader neutralization range isobtained.

Either a single vaccination or multiple vaccinations of subjects withthe peptide carrier conjugates may be used to induce the production ofhigh affinity/avidity neutralizing and/or protective HIV-specificantibodies. The preferred dose range is 50-500 mg of peptide perconjugate. At a later date, antibody-containing fluid is extracted fromthe vaccinated subjects. The antibody-containing fluid is then assessedin an assay. The assay is an antigen-limited ELISA which selects forhigh affinity/avidity neutralizing and/or protective HIV-specificantibodies.

In order to produce this assay, microplate wells are covered with theantigen which the antibodies are reactive to, such as MN-PND, or anotherPND antigen derived from other seroprevalent HIV strains, in adecreasing coating concentration series as follows:

Row A—500 ng/ml

Row B—100 ng/ml

Row C—50 ng/ml

Row D—10 ng/ml

Row E—5 ng/ml

Row F—1 ng/ml

Row G—0.5 ng/ml

Row H—0 ng/ml

Where the vaccine comprises a cocktail of different peptides conjugatedto carriers, all of the wells in the plate are covered with all of thepeptides in the vaccine in order to determine whether there wasinduction of high affinity/avidity neutralizing and/or protectiveHIV-specific antibody production. If such antibodies were produced, asecond ELISA is performed, wherein each row of the plate is covered witha separate peptide from the cocktail.

Next, antibody-containing fluid is removed from the vaccinated subjects,diluted with antibody-containing fluid diluent to a ratio of about 1:21(sample: diluent) and added to the series of wells. Theantibody-containing fluid diluent comprises a formulation of additivesin a buffered solution containing thimerosal. An example of suchantibody-containing fluid diluent is 0.1-0.5% casein in PBS containing0.05% Tween-20 (pH 7.4), 0.001 % rhodamine and thimerosal. Anyantibody-containing fluid may be used. Examples of antibody-containingfluid are serum, plasma, cerebral fluids and mucosal fluids. A negativecontrol, such as anti-HIV negative human serum, and a positive control,such as anti-HIV positive human serum or anti HIV-MN-PND monoclonalantibody should also be added to wells in the ELISA plate. The plateshould then be covered and incubated for about 60 minutes at about 37°C.

After incubation, the plate should be washed 5-6 times using dilutedplate wash solution (phosphate buffered saline, pH 7.4 with 0.05%Tween-20 diluted with deionized water containing chloracetamide at1:10). Use of an automatic plate washer is recommended. After washing,100 ml per well of anti-human conjugate such as peroxidase-conjugated(goat) Fab1 anti-human IgG, anti-human IgA or anti-human secretorycomponent (SC), diluted to 1:100,000 in conjugate diluent is added tothe wells. Any conjugate diluent may be used. The conjugate diluent maycomprise, for example, a formulation of additives in a buffered solutionwhich is typically added to antibody conjugate solutions by thoseskilled in the art. Subsequently, the wells are incubated for 60 minutesat 37° C. and washed 5-6 times using the diluted plate wash solution.(Again, use of an automatic plate washer is recommended.)

After washing, 200 ml of substrate such as O-phenylenediamine (o-PD)tablets, diluted (to 1 tablet per 12 ml of diluent) with hydrogenperoxide in a citrate buffer is added per well. The wells are thenincubated in the dark for 30 minutes at room temperature. The reactionis then stopped by adding 50 ml per well of 4N sulfuric acid. The platemay then be read in a microplate spectrophotometer at an absorbance of492 nm. (Use of a 620 nm reference filter is recommended.)

For the controls, the human negative control values should averagearound 0.050. The cutoff should be around 0.100. The human positivecontrol should filter out at least to the fifth well (5 ng/ml).

For the antibody-containing fluid, if the absorbance value is greaterthan the cutoff in the fourth row or less, the antibody. in the fluid islow affinity. If the absorbance value is greater than the cutoff in thefifth row or more (5 ng/ml), the antibody is medium affinity. If theabsorbance value is greater than the cutoff in the fifth row or more(less than or equal to 5 ng/ml), the antibody is high affinity. Adisplacement assay may then be set up to ascertain the high affinityantibodies.

The affinity of the elicited antibodies (in vivo and in vitro) may befurther assessed using the PHARMACIA BIACORI System, which is abiosensor-based technology (Biospecific Interaction Analysis, (BIA)which assesses biomolecular interactions to the picomolar range withoutthe need for labels (radioactive or fluorescent). BIA measures the KAand kDa of molecules bound to a surface with molecules in freesolutions. By reflecting a beam of light on a miniature sensing surface,it is possible to obtain quantitative kinetic data aboutantigen/antibody binding (association-dissociation constraints).

Another method of determining whether antibodies are highaffinity/avidity is the use of an immunoassay wherein an indirect ELISAis prepared by coating a solid phase with one or more synthetic peptidesderived from one or more different HIV strains, wherein each peptidecomprises an antigen including HIV PND. On the solid phase, a series ofassays can be prepared wherein each peptide is coated at varyingconcentrations,. from the nanomole to the femtomole range. Theimmobilized peptide is then contacted with sera from the subjectsvaccinated with the conjugates of this invention. Bound antibodies aredetected by the addition of an enzyme labeled or gold labeledanti-species conjugate. The color generated is proportional to theconcentration of high affinity/avidity antibodies. Suboptimal orrate-limiting antibody coating concentrations are used to. select outneutralizing antibodies so that the ELISA results are correlated withthe result of virus neutralization.

The solid phase ELISA can also utilize a secondary antibody againsthuman secretory component or IgA. This assay can then rapidly detectprotective antibodies in mucosal secretions such as saliva orcervicovaginal fluid, or serum IgA antibodies. Since IgA does not crossthe placenta, the presence of such antibodies in spinal cord bloodallows for the early detection of fetal HIV infection.

In order to achieve maximal assay sensitivity, antigen-coatingconcentrations to be applied to the solid phase ELISA should representthe minimal antigen concentration which is on the upper plateau, i.e.,maximum assay response. This area represents the maximal separation ofspecific from non-specific signals. Coating concentrations in the lowend of the titration curve represent an insensitive assay since a largeproportion of an individual's immune response would be missed.

As discussed above, suboptimal antigen concentrations may be utilized toeliminate weak antigen-antibody interactions. For example, it isgenerally understood that the coating of solid surfaces by antigensinvolve titrating the antigen for reactivity using a standard antiserum.Such titrations typically yield a sigmoidal-shaped curve of assayresponse. At high antigen coating concentrations which saturate thesolid phase, a maximal assay response will occur. This is seen as a“plateau”, a region of the titration curve which shows little change inthe amount of antibody detected regardless of the change in the amountof antigen. As antigen concentrations are lowered to non-saturating or“suboptimal” levels, assay responses decrease proportionally. Hence, anantigen-coating concentration which represents the minimal antigenconcentration which is on the upper plateau should be utilized in thesolid phase ELISA of this invention.

The conjugate vaccines which have induced the production of highaffinity/avidity neutralizing and/or protective HIV-specific antibodies,such antibodies being selected for by the ELISA, are “copied”. The“copies” are then used in the treatment and transmission prevention ofHIV.

EXAMPLE I

Immunization of Mice with Conjugates of Peptides Coupled to CarriersKLH, PPD, Avidin and Tetanus

Mice were immunized with peptide KRIHIGPGRAFYT (SEQ ID NO:1) (MN peptideor MN-PND) coupled to different carriers. Five mice were immunized withthe MN peptide coupled to Keyhole Limpet Hemocyanin (KLH) as describedin Methods Enzyol., 70:159 (1980). The mice were bled and antibodytiters directed against MN-PND were determined by an ELISA specific forMN-PND. Serum at a 1:1400 dilution obtained from mice immunized with theKLH-MN-PND conjugate had optical densities of 3.85 (K0), 0.598 (K1),1.762 (K2), 0.282 (K3), and 0.356 (K4) with a background of less than0.1. Four months later, the mice were boosted with KLH-MN-PND and thenthe sera was assayed for reactivity. This data is outlined in Table 1.All of the mice immunized had high titers and high affinity antibodiesto MN-PND. Mouse K4 was sacrificed. Its spleen was harvested and used todevelop monoclonal antibodies to MN-PND. Monoclonal antibodiesrecognizing MN-PND were generated, some of which were neutralizing.

TABLE 1 Reactivity of mice immunized with MN-PND coupled to KLH MN-PNDCONTROL KO KI K2 K3 K4 (ng/ml) 4/11/90 4/20/90 4/11/90 4/20/90 4/11/904/20/90 4/11/90 4/20/90 4/11/90 4/20/90 4/11/90 10000  0.06  0.058 0.0992.277 0.604 2.26  0.145 0.89  0.096 2.714 0.166 5000 0.054 0.056 0.1511.946 0.173 2.098 0.076 0.279 0.075 1.704 0.108 1000 0.093 0.056 0.0731.096  0.1312 1.79  0.125 0.405 0.075 2.054 0.165  500 0.094 0.056 0.0731.231 0.105 1.23  0.111 0.268 0.101 1.729 0.135  100 0.055 0.053 0.07 0.229 0.069 0.423 0.084 0.149 0.091 1.018 0.118  50 0.062 0.057 0.1120.477 0.108 0.389 0.107 0.254 0.094 0.748 0.144  10 0.059 0.059 0.0830.14  0.094 0.239 0.45  0.093 0.105 0.578 0.136   1 0.061 0.057 0.0740.11  0.091 0.091 0.171 0.099 0.078 0.261 0.161

EXAMPLE II

Immunization of Guinea Pigs with Conjugates of Peptides Coupled toCarrier Toxin A

Production of PND-Toxin A Conjugate. One gram of solid carbodiimide wasadded to 100 mg of toxin A derivatized with adipic acid dehydrazide(TA-ADH, 5.9 mg/ml in phosphate buffered saline, pH 7.4; PBS). The pHwas adjusted to 4.8 by the addition of 0.3N HCl. To this mixture 50 mgof MN-PND peptide in 2.5 ml of pyrogen-free water was slowly added underconstant stirring. The mixture was stirred for 3 hours at ambienttemperature. The mixture was filter sterilized using a 0.45 mm filterunit and passed through a Sephadex G75 column to separate the conjugatefrom reactants. The conjugate-containing fraction which eluted in thevoid volume was concentrated, dialyzed against PBS and filtersterilized. The conjugate was diluted to 200 mg total protein/ml in PBSand absorbed into A1(OH)₃.

Immunization Studies. Three guinea pigs were vaccinated intramuscularlyon days 0 and 14 with 50 mg of absorbed conjugate. Serum samples wereobtained on days 0, 14 and 28. An anti-MN-PND peptide ELISA wasperformed by coating plates with MN-PND peptide, reacting wells withserial dilutions of antisera, and after washing, reacting plates with ananti-guinea pig IgG antibody. As shown in Table 2 below, this conjugatedid not stimulate an anti-MN-PND antibody response.

TABLE 2 Day mean ELISA titer 0 1,4 14 1,8 28 1,0

EXAMPLE III

Immunization of Mice and Guinea Pigs with Conjugates of Peptides Coupledto Carrier Toxin A

Mice and guinea pigs were immunized with MN-PND coupled to Pseudomonasaeruginosa exotoxin A (toxin A) with a ratio of peptide to carrier of3:1. In addition, the MN-PND lacked a tyrosine residue next to thecarboxy terminus. The animals were immunized on days 0 and 14. Sera wasobtained at days 0, 14, and 28. The doses used were 5 and 25 mg in theguinea pigs and 2 and 10 mg in the mice. The immunizations were given aseither conjugate alone or in alum. None of the sera obtained from theseimmunizations had any reactivity to MN-PND. This was probably due toeither the immunogenicity of toxin A, coupling problems or the dosageused. Table 3, below, shows the ability of sera from guinea pigsimmunized with MN-PND-toxin A+PPD to neutralize the MN strain of HIV.The animals were immunized at days 0 and 14. Vales are for seracollected on day 28.

TABLE 3 GUINEA PIG SERA Serum Dilution 1:25 1:50 1:100 1:200 HIV-MNControl 115,030 130,400 126,400 135,030 Control Serum 84,890 86,69085,860 90,630 82 14 940 (98.9%) 1,123 (98.8%) 1,891 (97.8%) 2,171(97.7%) 1:20 titer 82 17 3,990 (95.3%) 80,700 (7.0%) 85,590 (0.4%)90,610 (0.1%) 1:20 titer 82 18 866 (99.0%) 1,079 (98.8%) 1,067 (96.8%)1,538 (98.4%) 1:200 titer 82 19 2,999 (96.5%) 5,098 (94.2%) 13,110(77.8%) 28,070 (69.1%) 82 20 2,589 (97.0%) 2,847 (96.8%) 2,804 (96.8%)5,111 (91.4%) Percentage calculated on Control Serum

EXAMPLE IV

Immunization of Guinea Pigs with Conjugates of Peptides Coupled toCarrier PPD

Preparation of PPD-MN-PND Conjugate. 4 mg of MN-PND in 400 ml of steriledistilled H₂O was mixed with 4 ml of sterile PPD-RT23 (1 mg/ml). CarrierPPD-RT23 was obtained from Statens Serum Institute, Copenhagen, Denmark.Carrier PPD-298 was obtained from Connaught Laboratories, Willowdale,Toronto. PPD-298-H2O signifies that the conjugation of the PND-MNpeptide and the PPD-298 carrier was performed in water. PPD-298-PBSsignifies that the conjugation of the PND-MN peptide and the PPD-298carrier was performed in PBS. 45 ml of 0.2% glutaraldehyde was thenadded to the PPD-MN-PND solution, mixed by vortexing and incubated inthe dark at 22° C. for 30 min. An additional 20 ml of 0.2%glutaraldehyde was added and the solution was incubated for 90 min. Thesolution appeared opalescent at this time. The reaction mixture wastransferred aseptically into a sterile dialysis tubing and dialyzedagainst 1 liter of sterile PBS at 4° C. for 24 hours. The concentrationof PPD was calculated by dividing the total amount of PPD added to thereaction mixture by the final volume of the sample after dialysis. Analiquot of the conjugate was diluted in sterile pyrogen-free PBS to 20mg of PPD per ml (0.1000 E/ml) for determining the sterility and generalsafety of the vaccine preparation according to the guidelines ofEuropean Pharmacopeia. The remainder of the sample was kept in undilutedform at 4° C.

Given the heterogeneous nature of PPD, it was not possible to accuratelydetermine the ratio of PPD: MN-PND conjugate by amino acid analysis.Therefore, incorporation of ¹²⁵I MN-PND peptide into PPD in the presenceof glutaraldehyde was determined.

A MN-PND (10 mg in 0.2 ml H₂O) solution was prepared. A ¹²⁵I (1 mCidiluted with 10 ml of 0.1 N NaOH) solution was prepared. Ten (10) ml of¹²⁵I solution was added to 0.2 ml MN-PND solution. To this 80 mlchloramine T (2 mg/ml in 0.1 M NaPO₄, pH 7.2) was added. The solutionwas mixed for 1 minute at room temperature and immediately applied to aSephadex G-10 column equilibrated in PBS, pH 7.4. Fractions (30 sec)were collected. The radioactivity per fraction was determined and thepeak fractions collected and pooled. The total MN-PND peptide present inthe pool was calculated by Lowry using the Folin reagent. Aconcentration of 3.23 mg/ml was yielded. The total 125I MN-PND was68,348 CPM/mg MN-PND.

Estimation of the ¹²⁵I MN-PND incorporation into PPD was determined asfollows: To 2 ml PBS, pH 7.4, were added 2 mg PPD together with 2 mg¹²⁵I MN-PND. Coupling was performed by the addition of glutaraldehyde.After coupling, the conjugate was extensively dialyzed against PBS, pH7.4. The conjugate was removed from the dialysis tubing. A total of710,000 CPM/2 mg PPD was incorporated, corresponding to 1.29 mg MN-PND.Total incorporation of the MN-PND peptide was roughly 59% (710,000 CPMof total 1,200,000 CPM added). Therefore, 2 mg of PPD contained 1.29 mgof MN-PND, assuming total recovery of PPD. The ratio of PPD to MN-PNDwas 1:0.645. (A human dose of 50 IU of PPD would contain approximately0.645 mg of MN-PND.)

Immunogenicity of PPD-MN-PND Conjugate in Guinea Pigs. Groups of 5guinea pigs were primed with 10⁶ CFU of BCG or saline. The animals werethen immunized with 10 or 50 mg of PPD-MN-PND conjugate in 0.1 ml PBS 14and 28 days after priming. The animals were bled at 14, 28 and 42 daysafter priming. The sera was then assayed in an MN-PND ELISA to determineimmunogenicity of the PPD-MN-PND conjugate.

Protocol for MN-PND HIV ELISA. Dynatech polystyrene plates were coatedwith 100 ml of a 1.0 mg/ml of MN-PND (in carbonate buffer, pH 9.6) at22° C. overnight. The solution was removed and then blocked with 100 mlof a 1 mg/ml solution of Bovine Serum Albumin (BSA) in PBS (pH 7.4) for1 hour at 22° C. A 100 ml of 2-fold serial dilutions of sera inPBS-tween (ph 7.4) starting at 1:20 were dispensed into each well andthe plates incubated for 3 hours. The plates were washed 3 times with300 ml of PBS-TWEEN. 100 ml of peroxidase-conjugated rabbit anti-guineapig IgG (1:2500 in PBS TWEEN, Nordic Immunology, Tilburg, TheNetherlands) was added to each well and the plates were incubated for 2hours. The plates were washed three times in PBS-TWEEN and 100 ml ofp-nitrophenyl phosphate (Merck) was added to the wells. The plates werethen incubated for 60 minutes at 22° C. The absorbance at 405 nm wasmeasured using a Dynatech MR5000 Microtiter Plate Reader (Dynatech,Switzerland). The serum dilution at the linear part of the titrationcurve (between 0.15 and 0.6) was multiplied by the reciprocalcorresponding dilution of the serum and expressed as ELISA units.

As shown in Table 4 below, priming of the guinea pigs with BCG andsubsequent immunization with the PPD-MN-PND conjugate induced theproduction of high affinity/avidity neutralizing and/or protectiveHIV-specific antibodies. The antibody titers of the guinea pigs primedwith BCG were much higher than the titers of the guinea pigs not primedwith BCG, indicating that priming with BCG prior to conjugateimmunization causes the immune system to respond better to conjugateimmunization, thereby more effectively inducing the production of highaffinity/avidity neutralizing and/or protective HIV-specific antibodies.The antibody titers of the guinea pigs not primed with BCG (group d)were not statistically higher than guinea pigs not immunized with thePPD-MN-PND conjugate vaccine (group a).

TABLE 4 Immunogenicity of PND-MN-PPD Conjugates in BCG Primed andUnprimed Guinea Pigs Geometric Mean Vaccine Dose (μg) Elisa unit (range)— — 1.6 (1.2-2.7)^(a) PPD-MN-PND 10 2.4 (2.14-2.78)^(d) PPD-MN-PND 506.0 (5.68-6.12)^(d) PPD-MN-PND 10 121.1 (5.40-462.1)^(b) PPD-MN-PND 5014.1 (5.56-25.68)^(c) RT23 10 21.0 (6.7-70.8)^(b) RT23 50 180.5(49.4-692.5)^(b) PPD-298-H20 10 31.1 (4.9-181.9)^(b) PPD-298-H20 50142.2 (31.0-393.0)^(c) PPD-298-PBS 10 22.8 (6.3-232.0)^(c) PPD-298-PBS50 63.2 (9.6-858.9)^(c) ^(a)Geometric Mean Elisa units of Pre immunesera from 4 guinea pigs. ^(b)Geometric Mean Elisa units of post immunesera from 4 guinea pigs. ^(c)Geometric Mean Elisa units of post immunesera from 3 guinea pigs. ^(d)Guinea pigs not primed with BCG.

EXAMPLE V

Immunization of Guinea Pigs and Mice with Conjugates of Peptides Coupledto Carrier PPD

Guinea pigs and mice were immunized with MN-PND coupled to 2 types ofPPD. Some of the PPD-immunized animals were first primed with BCG. Table5 shows the immunoreactivity of the animals to MN-PND. Table 6 shows thetitration of serum reactivity to MN-PND. Table 7 shows the affinity toMN-PND as measured by antigen-limited ELISA. Samples were assayed forneutralization activity. Results obtained indicated that neither of thesera had neutralizing activity to MN-HIV.

TABLE 5 Reactivity of Mice and Guinea Pigs immunized with MN-PPD coupledto PPD GUINEA PIG MICE SAMPLE DAY O.D. SAMPLE DAY O.D.   0  0 0.15   0 0 0.119 1/2/3/4  0 0.01 MN-PND-PPD 10 ug 7281 28 0.14 MD-PND-PPD 2 ugGROUP #1 28 0.026 7282 28 0.62 GROUP #1 28 0.018 7284 28 0.312 GROUP #128 0.023 7285 28 0.43 MN-PND-PPD 10 ug GROUP #2 28 0.026 MN-PND-PPD 50ug 7286 28 2.4 GROUP #2 28 0.02 7287 28 2.9 GROUP #2 28 0.043 7288 281.448 GROUP #2 28 0.02 BCG/MN-PND-PPD 7291 42 3.6 BCG/MN-PND-PPD GROUP#3 28 0.026 10 ug 2 ug 7292 28 0.27 GROUP #3 42 0.075 7293 42 3.6 GROUP#3 42 0.469 7294 42 3.1 GROUP #3 42 1.7 7295 42 3.3 GROUP #3 42 2.4BCG/MN-PND-PPD 7296 42 3.39 BCG/MN-PND-PPD GROUP #4 28 0.098 50 ug 10 ug7298 42 3.5 GROUP #4 42 1.3 7299 0.275 GROUP #4 42 2.1 7300 42 3.96

TABLE 6 Titration of guinea pig anti-MN-PND Serum DILUTION SAMPLE DAY1:20 1:100 1:500 1:1000 1:2500   0  0 0.15 0.07 0.05 0.05 0.04MN-PND-PPD 50 ug 7286 28 2.40 0.36 0.09 0.07 0.05 7287 28 2.90 0.40 0.120.09 0.07 7288 28 1.45 0.17 0.07 0.08 0.06 BCG/MN-PND-PPD 10 ug 7291 423.60 3.71 3.67 3.30 1.61 7292 28 0.27 7293 42 3.60 0.52 0.12 0.08 0.077294 42 3.10 1.30 0.27 0.18 0.13 7295 42 3.10 1.50 0.03 0.18 0.13BCG/MN-PND-PPD 50 ug 7296 42 3.39 1.17 0.27 0.15 0.10 7298 42 3.50 0.240.09 0.06 0.04 7299 0.28 7300 42 3.96 3.76 3.81 2.48 0.95

TABLE 7 Antigen limited MN-PND ELISA of guinea pig samples IMMUNIZATIONMN-PND HN-PND-PPD 50 ug BCG/HIV-PPD-PND 10 ug BCG/MN-PND-PPD 50 ug(ng/well) SAMPLE 0 7286 7287 7288 7291 7293 7294 7295 7296 7298 7300 5000.21 2.13 2.84 0.98 3.95 3.64 3.99 3.99 3.49 3.50 3.84 100 0.32 2.472.89 1.08 3.50 3.30 3.53 3.59 3.60 3.21 3.75 50 0.31 1.97 2.49 0.77 3.963.08 3.53 3.46 3.41 2.18 3.82 10 0.22 0.86 0.63 0.50 3.96 1.35 1.16 2.600.82 0.52 3.01 5 0.26 0.15 0.37 0.36 3.53 0.46 0.56 1.59 0.44 0.29 1.281 0.21 0.18 0.26 0.30 0.73 0.31 0.25 0.29 0.25 0.24 0.24 0.5 0.18 0.160.23 0.24 0.72 0.33 0.21 0.24 0.23 0.22 0.22

EXAMPLE VI

Immunization of Humans with Conjugates of Peptides Coupled to CarrierPPD

Based on the immunogenicity of the PPD-MN-PND conjugates in guinea pigsillustrated in EXAMPLE V, five PPD-positive humans were immunized withthis conjugate. The reactivity of sera from the 5 human volunteers wasassayed one month after immunization with PPD-MN-PND. No significant IgGor IgM to MN-PND was detected. The sera was again assayed two monthsafter immunization. Table 8 shows the results of the assay.

TABLE 8 Reactivity of human volunteers to MN-PPD-PND 2 months afterimmunization Specific Absorbance (410 nm) Negative Control 0.067 0.0540.063 Negative Control 0.099 0.059 0.096 Positive Control 0.3 0.4340.357 Volunteer #1 0.229 0.463 0.388 Volunteer #2 0.054 0.068 0.068Volunteer #3 0.096 0.114 0.105 Volunteer #4 0.055 0.065 0.071 Volunteer#5 0.049 0.055 0.076

The volunteers were immunized a second time. Serum samples were obtainedabout 14 days and 28 days after each immunization and prior toimmunization. As shown in Table 9 below, after the second immunization,one subject was a very strong responder, one was a “borderline”responder and 2 were non-responders.

TABLE 9 Assay for reactivity to MN-PND was performed in triplicate onthe samples obtained 44 days after immunization. Sample #1 .370 ± .057Sample #2 .063 ± .038 Sample #3 .105 ± .004 Sample #4 .063 ± .004Positive .363 ± .031 Negative #1 .084 ± .010 Negative #2 .061 ± .003Sample #1 is significantly different from negative controls (p < .001)and sample #3 may be significantly different from negative control (p <.05).

The volunteers were immunized a third time. After the third immunizationwith the PPD-MN-PND conjugate, the serum of one volunteer had a hightiter of high affinity/avidity HIV-specific antibodies. Upon exposure toMN-PND, the lymphocytes responded in vitro by proliferation andsecretion of interleukin-2. This shows that an entire immune responsewas induced. B-cell humoral immunity response was induced, as evidencedby the production of antibodies. T-cell immunity response was alsoinduced, as shown by the proliferation of T-cells upon exposure to theconjugate, and as shown by the secretion of interleukin-2, which is amediator of the immune response. The other three serum samples showed nosignificant immunological response. The fact that high affinity/avidityHIV-specific antibodies are found more frequently in HIV infectedasymptomatic subjects indicates that such antibodies are protective.

EXAMPLE VII

Synthesis of Peptides and Immunization of Mice with Conjugates ofSynthesized Peptides Coupled to Carrier KLH

In addition to MN-PND, four other peptides were synthesized. Thesepeptides are 282 (GPGRAFGPGRAFGPGRAFC) (SEQ ID NO:5), 283 (IYIGPGRAC)(SEQ ID NO:2), 284 (IAIGPGRAC)(SEQ ID NO:3) and 285 (IHIGPGRAC) (SEQ IDNO:4). These peptides were coupled to KLH as described above. Five micewere immunized with each peptide conjugate and boosted 2 months later.Serum was obtained. FIG. 1 shows the reactivity to each of the peptides.FIG. 2 shows the affinity/avidity of the mice with the highest titers ofantibody. There was variable immunogenicity of the peptides. 284 was themost highly immunogenic, and 285 was not immunogenic at all. Some miceimmunized with 283 and 284 had very low reactivity to MN-PND. This wasnot just an ELISA artifact as evidenced by the fact that when peptide282 was incubated with serum from mouse 283-5 prior to performing theELISA, peptide 282 bound to (competed with) the antibodies in the mouseserum, whereas the MN peptide did not compete with the mouse serumantibodies when incubated therewith. In contrast, peptides 282 and MNboth were able to compete with (bind to) the antibodies in the serum ofmouse 284-4 (see FIG. 3).

EXAMPLE VIII

Immunoreactivity of Humans to Synthesized Peptides

Based on the observation of the differential immunogenicity of the PNDpeptides, sera from 20 HIV-positive subjects was assessed for reactivitywith the five indicated peptides. (See Table 10). Eight samples reactedwith at least 4 out of 5 peptides, one sample. with 3 out of 5, onesample with 2 out of 5 peptides, and six samples with only 1 out of 5peptides. One sample was negative, and three samples had highbackgrounds. Of eight subjects who had high affinity antibodies toMN-PND, two subjects had high affinity antibodies to 282. In addition,of the eight subjects who had high affinity antibodies to MN-PND, 5recognized 5out of 5 peptides, one recognized 4 out of 5 peptides, 1recognized 3 out of 5 peptides and 1 recognized 1 out of 5 peptides.This indicates that within the group of subjects with highaffinity/avidity antibodies to MN-PND, there exist subgroups, some ofwhich may be more protective against HIV. In addition this may haveclinical prognostic significance which will affect the inclusion ofpeptides in a multivalent vaccine.

TABLE 10 Affinity/Avidity CONCENTRATION (ng/ml) SAMPLE PEPTIDEReactivity +/− (ng/ml) 5000 1000 500 100 50 10 5 0 NegativeKRIHIGPGRAFYT (SEQ ID NO: 1) 0.014 ″ 0.018 (GPGRAF)(SEQ ID NO: 16) 3C0.02 ″ 0.035 IYIGPGRAC (SEQ ID NO: 2) 0.03 ″ 0.018 IAIGPGRAC (SEQ ID NO:3) 0.025 ″ 0.021 IHIGPGRAC (SEQ ID NO: 4) 0.033 ″ 0.019 2207KRIHIGPGRAFYT + 2.949 3.089 2.909 2.426 1.828 0.857 0.608 0.469(GPGRAF)3C + 1.544 0.579 1.123 0.607 0.861 0.486 0.617 0.775 IYIGPGRAC +2.884 2.444 2.544 0.422 0.787 0.496 0.776 0.711 IAIGPGRAC + 3.039 2.781.985 0.484 0.894 0.677 0.832 0.965 IHIGPGRAC + 3.126 2.981 2.789 0.7731.047 0.634 1.115 1.083 2434 KRIHIGPGRAFYT + 1.808 1.517 0.828 0.298 0.30.272 0.178 0.324 (GPGRAF)3C + 3.392 2.979 2.857 2.03 1.899 0.097 0.8421.14 IYIGPGRAC + 1.661 1.425 1.32 0.184 0.746 0.358 0.628 0.96IAIGPGRAC + 3.128 2.872 2.028 0.41 0.677 0.355 0.32 0.859 IHIGPGRAC +2.136 1.913 1.512 0.55 0.367 0.334 0.632 0.453 2477 KRIHIGPGRAFYT + + 502.709 2.415 2.606 1.365 0.715 0.158 0.103 0.095 (GPGRAF)3C + + 50 3.22.137 2.212 0.323 0.385 0.041 0.074 0.152 IYIGPGRAC + − 1000 0.776 0.6880.1 0.055 0.021 0.027 0.053 0.091 IAIGPGRAC + − 500 2.366 0.856 0.9830.053 0.091 0.023 0.046 0.136 IHIGPGRAC + − 500 1.169 1.454 1.415 0.1380.115 0.022 0.045 0.155 2529 KRIHIGPGRAFYT + + 50 1.668 1.326 1.2840.358 0.223 0.059 0.061 0.084 (GPGRAF)3C + − 500 2.29 1.23 0.666 0.0380.049 0.04 0.052 0.108 IYIGPGRAC + − 1000 0.789 0.303 0.169 0.038 0.0510.031 0.047 0.079 IAIGPGRAC + − 1000 2.243 0.432 0.124 0.014 0.04 0.0360.047 0.073 IHIGPGRAC + − 500 1.214 2.023 1.35 0.073 0.063 0.055 0.0460.092 2234 KRIHIGPGRAFYT + + 10 3.049 2.932 2.547 1.705 1.334 0.2040.111 0.075 (GPGRAF)3C + − 500 0.564 0.482 0.303 0.038 0.039 0.028 0.050.042 IYIGPGRAC + − 5000 0.218 0.118 0.091 0.027 0.029 0.026 0.039 0.039IAIGPGRAC + − 1000 2.34 0.389 0.116 0.016 0.044 0.036 0.03 0.049IHIGPGRAC + − 500 2.361 0.525 1.042 0.023 0.049 0.033 0.048 0.057 2488KRIHIGPGRAFYT + + 1 2.983 3.131 3.012 2.367 1.777 0.531 0.307 0.241(GPGRAF)3C + + 50 1.282 0.983 0.91 0.536 0.43 0.088 0.131 0.144IYIGPGRAC + − 1000 0.269 0.226 0.155 0.092 0.052 0.079 0.095 0.126IAIGPGRAC + − 500 1.204 0.254 0.223 0.072 0.028 0.063 0.054 0.123IHIGPGRAC + − 500 2.968 2.829 2.338 0.135 0.129 0.077 0.1 0.115 2535KRIHIGPGRAFYT + − 500 0.584 0.26 0.237 0.162 0.124 0.08 0.182 0.075(GPGRAF)3C + − 500 2.244 1.274 0.685 0.071 0.079 0.076 0.075 0.082IYIGPGRAC − − — 0.115 0.091 0.077 0.07 0.07 0.06 0.086 0.099 IAIGPGRAC +− 5000 0.347 0.119 0.162 0.063 0.085 0.067 0.087 0.097 IHIGPGRAC + − 5002.244 1.943 1.339 0.141 0.1 0.144 0.08 0.094 2126 KRIHIGPGRAFYT + + 102.136 1.877 1.627 0.603 0.485 0.308 0.177 0.135 (GPGRAF)3C + − 500 0.6730.517 0.333 0.055 0.108 0.054 0.096 0.091 IYIGPGRAC + − 500 2.633 2.0491.857 0.077 0.14 0.037 0.089 0.053 IAIGPGRAC + − 5000 0.266 0.132 0.0990.026 0.03 0.025 0.041 0.09 IHIGPGRAC + − 500 2.964 2.612 2.099 0.1060.125 0.049 0.099 0.02 2146 KRIHIGPGRAFYT + − 100 0.3 0.238 0.221 0.2510.188 0.092 0.051 0.06 (GPGRAF)3C ? ? ? 0.055 0.096 0.072 0.22 0.3330.281 0.349 0.574 IYIGPGRAC ? ? ? 0.469 0.423 0.435 0.201 0.275 0.2580.28 0.489 IAIGPGRAC ? ? ? 0.525 0.255 0.278 0.24 0.296 0.231 0.2790.405 IHIGPGRAC ? ? ? 1.002 0.806 0.683 0.295 0.45 0.301 0.442 0.7 2115KRIHIGPGRAFYT + − 100 1.325 1.367 1.263 0.374 0.235 0.135 0.063 0.224(GPGRAF)3C + − 5000 0.254 0.13 0.11 0.071 0.042 0.084 0.092 0.135IYIGPGRAC + − 5000 0.237 0.16 0.254 0.037 0.042 0.045 0.046 0.07IAIGPGRAC + − 5000 0.783 0.143 0.11 0.06 0.065 0.062 0.091 0.12IHIGPGRAC + − 5000 0.346 0.187 0.166 0.058 0.07 0.058 0.081 0.168 2270KRIHIGPGRAFYT + − 5000 0.339 0.137 0.104 0.044 0.044 0.045 0.038 0.074(GPGRAF)3C + − 500 0.7 0.528 0.34 0.04 0.043 0.035 0.034 0.065 IYIGPGRAC− − — 0.096 0.074 0.052 0.022 0.026 0.026 0.026 0.033 IAIGPGRAC − − —0.089 0.096 0.12 0.056 0.071 0.043 0.075 0.073 IHIGPGRAC − − — 0.0620.071 0.076 0.061 0.08 0.053 0.08 0.101 2125 KRIHIGPGRAFYT + + 10 2.6832.704 2.652 1.748 1.416 0.244 0.124 0.063 (GPGRAF)3C + - 500 1.904 0.9470.905 0.046 0.045 0.049 0.071 0.107 IYIGPGRAC − − — 0.07 0.071 0.0790.043 0.067 0.036 0.068 0.069 AIAGPGRAC + − 5000 0.266 0.132 0.099 0.0260.03 0.025 0.041 0.039 IHIGPGRAC + − 5000 0.291 0.185 0.097 0.028 0.0380.031 0.034 0.031 2335 KRIHIGPGRAFYT + + 10 1.839 1.194 2.062 1.5331.186 0.313 0.174 0.1 (GPGRAF)3C + − 1000 0.426 0.349 0.183 0.032 0.050.021 0.068 0.126 IYIGPGRAC − − — 0.148 0.073 0.031 0.022 0.047 0.0260.055 0.098 IAIGPGRAC − − — 0.184 0.123 0.129 0.043 0.034 0.034 0.0430.098 IHIGPGRAC + − 5000 0.209 0.102 0.149 0.03 0.047 0.042 0.056 0.0882232 KRIHIGPGRAFYT + + 50 1.148 1.083 0.979 0.537 0.281 0.078 0.0650.075 (GPGRAF)3C − − — 0.12 0.064 0.053 0.03 0.041 0.025 0.037 0.047IYIGPGRAC − − — 0.044 0.046 0.024 0.019 0.028 0.017 0.026 0.047IAIGPGRAC − − — 0.063 0.055 0.043 0.021 0.034 0.027 0.033 0.059IHIGPGRAC − − — 0.158 0.033 0.048 0.027 0.035 0.027 0.035 0.045 2436KRIHIGPGRAFYT + − 500 1.306 0.98 0.579 0.093 0.057 0.025 0.026 0.022(GPGRAF)3C − − — 0.05 0.025 0.029 0.039 0.032 0.015 0.018 0.022IYIGPGRAC − − — 0.023 0.034 0.017 0.015 0.016 0.012 0.02 0.019 IAIGPGRAC− − — 0.033 0.019 0.021 0.023 0.016 0.024 0.026 0.018 IHIGPGRAC − − —0.084 0.051 0.031 0.018 0.015 0.016 0.02 0.023 2205 KRIHIGPGRAFYT − − —0.094 0.049 0.064 0.042 0.055 0.051 0.055 0.071 (GPGRAF)3C + − 500 1.0370.475 0.223 0.046 0.042 0.054 0.043 0.059 IYIGPGRAC − − — 0.113 0.0670.065 0.042 0.045 0.04 0.041 0.046 IAIGPGRAC − − — 0.111 0.034 0.0580.042 0.044 0.053 0.057 0.051 IHIGPGRAC − − — 0.143 0.105 0.072 0.0230.043 0.042 0.049 0.063 2236 KRIHIGPGRAFYT − − — 0.035 0.026 0.025 0.0280.032 0.024 0.028 0.032 (GPGRAF)3C − − — 0.141 0.079 0.057 0.016 0.0210.019 0.025 0.038 IYIGPGRAC − − — 0.043 0.031 0.032 0.017 0.017 0.0210.016 0.02 IAIGPGRAC + − 1000 2.335 0.914 0.137 0.018 0.021 0.023 0.0320.033 IHIGPGRAC − − — 0.095 0.049 0.04 0.019 0.023 0.024 0.033 0.04 2128KRIHIGPGRAFYT − − — 0.173 0.158 0.113 0.066 0.058 0.056 0.051 0.057(GPGRAF)3C − − — 0.196 0.119 0.08 0.029 0.035 0.031 0.043 0.045IYIGPGRAC − − — 0.125 0.087 0.072 0.026 0.026 0.021 0.026 0.049IAIGPGRAC − − — 0.078 0.034 0.044 0.025 0.034 0.027 0.019 0.049IHIGPGRAC + − 500 1.824 1.262 1.456 0.059 0.073 0.026 0.036 0.071 2332KRIHIGPGRAFYT − − — 0.043 0.04 0.029 0.034 0.031 0.028 0.025 0.028(GPGRAF)3C − − — 0.087 0.048 0.04 0.021 0.026 0.022 0.028 0.051IYIGPGRAC − − — 0.024 0.026 0.031 0.022 0.023 0.021 0.025 0.034IAIGPGRAC − − — 0.03 0.04 0.041 0.027 0.024 0.025 0.033 0.038 IHIGPGRAC− − — 0.036 0.039 0.032 0.026 0.027 0.021 0.026 0.048 2437 KRIHIGPGRAFYT− − — 0.059 0.052 0.117 0.121 0.036 0.036 0.125 0.098 (GPGRAF)3C + −1000 0.261 0.138 0.115 0.049 0.042 0.035 0.045 0.066 IYIGPGRAC − − —0.032 0.09 0.077 0.036 0.044 0.035 0.054 0.07 IAIGPGRAC − − — 0.0570.043 0.046 0.036 0.062 0.047 0.053 0.078 IHIGPGRAC − − — 0.103 0.0870.075 0.042 0.051 0.04 0.05 0.07

As shown in Table 11, Sample #2234 bound to MN-PND when incubated withMN-PND prior to ELISA, but not with peptide 282. Peptide 282 comprises(GPGRAF) (SEQ ID NO:16) 3C, and the MN peptide contains GPGRAF (SEQ IDNO:16) enclosed by two flanking sequences (KRIHIGPGRAFYT) (SEQ ID NO:1).This implies that the bulk of reactivity to MN-PND is to the flankingsequences. The inability of Sample #2232 to bind to peptide 282 impliesthat there is only reactivity to flanking sequences and not to GPGRAF.Sample #2434 is difficult to interpret because of high background.Sample #2270 has strong reactivity with peptide 282 in a conjugate, butno reactivity with soluble peptide 282. This suggests the possibilitythat adhered peptide 282 may express an epitope recognized by the serathat is not present in soluble peptide 282. Another possibility is thatthere is a discordance between affinity measured by the antigen-limitedELISA and that measured by competition.

TABLE 11 CONCENTRATION (ng/ml) SAMPLE PEPTIDE plate 5000 1000 500 100 5010 5 1 0 2234  0 MN 1.803 2.597 2.201 1.416 0.679 0.118 0.091 0.048 MN0.28 0.437 0.335 0.061 0.056 0.036 0.047 0.031 282 1.887 2.191 1.96 1.510.795 0.139 0.109 0.046  0 282 0.704 0.567 0.469 0.182 0.163 0.077 0.0860.085 282 0.222 0.152 0.106 0.058 0.05 0.091 0.04 0.07 MN 0.116 0.0570.125 0.069 0.068 0.058 0.074 0.073 2232  0 MN 1.352 1.36 1.159 0.7880.434 0.114 0.092 0.088 MN 0.952 0.927 0.727 0.286 0.15 0.069 0.0680.089 282 1.507 1.45 1.309 0.824 0.433 0.267 0.08 0.1  0 282 0.131 1.1040.106 0.085 0.097 0.086 0.074 0.053 282 0.124 0.103 0.103 0.083 0.1080.093 0.074 0.084 MN 0.114 0.083 0.097 0.107 0.091 0.099 0.098 0.09 2434 0 MN 1.14 0.584 0.669 0.173 0.254 0.311 0.202 0.282 MN 1.029 0.9260.488 0.294 0.745 0.408 0.341 0.392 282 0.644 0.598 0.35 0.234 0.2660.332 0.287 0.372  0 282 3.133 3.144 2.959 1.544 0.699 0.693 0.611 0.361282 3.238 3.117 2.356 0.666 0.405 0.654 0.598 0.556 MN 3.375 3.317 3.0521.626 1.021 0.754 0.55 0.588 2270  0 MN 0.07 0.095 0.066 0.044 0.0560.071 0.071 0.051 MN 0.097 0.076 0.105 0.055 0.05 0.089 0.058 0.097 2820.103 0.105 0.087 0.051 0.066 0.086 0.059 0.093  0 282 1.506 0.778 0.1410.05 0.087 0.083 0.082 0.073 282 1.458 0.809 0.163 0.07 0.088 0.0730.081 0.068 MN 1.313 0.838 0.159 0.088 0.069 0.079 0.077 0.066

EXAMPLE IX

Immunization of Humans with Conjugates of MN-PND Coupled to Carrier PPD

Twenty four subjects 23-63 years of age were tested to determine if theywere PPD-positive by Mantoux skin testing for immune response at 2 IEdosage. Those who were skin test positive were not further tested byMantoux. Those who were skin test negative were further Mantoux testedat 10 IE dosage. A total of 12 subjects were classified as PPD-positive.All twenty-four subjects were also tested by a third generation ELISAtest (Abbott Envacor) to confirm that they were not HIV antibodypositive. After Mantoux and HIV testing, twenty-three of the twenty-foursubjects were intradermally immunized with 40-50 IU conjugates of the MNpeptide (KRIHIGPGRAFYT) (SEQ ID NO:1) covalently coupled to the carrierPPD. These immunizations were performed at days 0, 14 and 28 on alltwenty-three subjects. A fourth immunization (booster dose) wasperformed on all of the PPD-positive subjects more than three months butless than five months after day 0. Blood was drawn between and after theimmunizations to determine whether the subjects produced anti-MN peptideantibody, whether this antibody possessed high affinity/avidity or wasable to neutralize HIV. Table 12 shows the dates of birth of eachsubject, the results of Mantoux and HIV testing for each subject, andprovides the immunization dates and blood withdrawal dates for thesubjects.

TABLE 12 Progress Control Immunization #1 Immunization #2 Immunization#3 Date of Mantoux Labor-/ Mantoux Day 0 Day 14 Day 28 Birth 2 IE/ResultHIV-Test 10 IE/Result Date Dose Date Dose Date Dose 08.24.46 01.27.92neg 01.30.92 — drop out 05.11.46 01.27.92 neg 01.30.92 — 02.05.92 2 × 25IE 02.19.92 2 × 20 IE 03.04.92 2 × 20 IE 11.13.54 01.27.92 pos 01.30.92— 02.10.92 2 × 25 IE 02.24.92 2 × 20 IE 03.09.92 2 × 20 IE 08.12.2701.27.92 pos 01.30.92 — 02.07.92 2 × 25 IE 02.21.92 2 × 20 IE 03.06.92 2× 20 IE 10.13.50 01.27.92 neg 01.30.92 01.30.92 neg 02.05.92 1 × 100 IE02.19.92 1 × 100 IE 03.04.92 1 × 100 IE 02.02.33 01.27.92 neg 01.30.9201.30.92 neg 02.05.92 1 × 100 IE 02.19.92 1 × 100 IE 03.04.92 1 × 100 IE11.06.35 01.27.92 pos 01.30.92 — 02.07.92 2 × 25 IE 02.20.92 2 × 20 IE03.05.92 2 × 20 IE 10.05.66 01.31.92 neg 01.30.92 02.93.92 pos 02.07.922 × 25 IE 02.21.92 2 × 20 IE 03.06.92 2 × 20 IE 03.20.37 01.27.92 pos01.30.92 — 02.07.92 2 × 25 IE 02.21.92 2 × 20 IE 03.06.92 2 × 20 IE04.28.71 01.27.92 neg 01.30.92 — 02.05.92 2 × 25 IE 02.19.92 2 × 20 IE03.04.92 2 × 20 IE 10.17.51 01.27.92 pos 01.30.92 — 02.07.92 2 × 25 IE02.21.92 2 × 20 IE 03.06.92 2 × 20 IE 03.28.56 01.27.92 neg 01.30.92 —drop out 03.10.50 01.31.92 neg 01.31.92 02.03.92 neg 02.07.92 1 × 100 IE02.24.92 1 × 100 IE 03.06.92 1 × 100 IE 10.09.48 01.28.92 neg 01.31.92 —02.05.92 2 × 25 IE 02.19.92 2 × 20 IE 03.04.92 2 × 20 IE 02.09.3101.28.92 pos 01.31.92 — 02.07.92 2 × 25 IE 02.21.92 2 × 20 IE 03.06.92 2× 20 IE 10.07.58 01.28.92 neg 01.31.92 01.30.92 neg 02.05.92 1 × 100 IE02.19.92 1 × 100 IE 03.04.92 1 × 100 IE 12.30.28 01.28.92 pos 01.31.92 —02.07.92 2 × 25 IE 02.21.92 2 × 20 IE 03.06.92 2 × 20 IE 04.21.3001.28.92 pos 01.31.92 — 02.07.92 2 × 25 IE 02.21.92 2 × 20 IE 03.06.92 2× 20 IE 09.15.41 01.28.92 pos 01.31.92 — 02.07.92 2 × 25 IE 02.24.92 2 ×20 IE 03.09.92 2 × 20 IE 01.20.42 01.28.92 pos 01.31.92 — 02.07.92 2 ×25 IE 02.21.92 2 × 20 IE 03.06.92 2 × 20 IE 01.16.47 01.28.92 pos01.31.92 — 02.07.92 2 × 25 IE 02.21.92 2 × 20 IE 03.06.92 2 × 20 IE05.09.51 01.28.92 neg 01.31.92 01.30.92 pos 02.05.92 2 × 25 IE 02.19.922 × 20 IE 03.04.92 2 × 20 IE 01.15.14 01.28.92 pos 01.31.92 — 02.07.92 2× 25 IE 02.21.92 2 × 20 IE 03.06.92 2 × 20 IE 05.07.44 02.10.92 neg02.13.92 02.10.92 neg 02.17.92 2 × 25 IE 03.02.92 2 × 20 IE 03.16.92 2 ×20 IE Blood Blood Date of Day 42 Immunization #4 sample sample BirthDate Date Dose #1 #2 08.24.46 05.11.46 03.18.92 11.13.54 02.23.925.19.92 2 × 20 IE 05.25.92 06.09.92 08.12.27 03.20.92 5.19.92 2 × 20 IE05.25.92 06.15.92 10.13.50 03.18.92 02.02.33 03.18.92 11.06.35 03.19.925.19.92 2 × 20 IE 05.25.92 06.09.92 10.05.66 03.20.92 03.20.37 03.20.925.19.92 2 × 20 IE 05.25.92 06.09.92 04.28.71 03.18.92 10.17.51 03.20.925.19.92 2 × 20 IE 05.25.92 06.09.92 03.28.56 03.10.50 03.23.92 10.09.4803.18.92 02.09.31 03.20.92 10.07.58 03.18.92 12.30.28 03.20.92 5.19.92 2× 20 IE 05.25.92 06.09.92 04.21.30 03.20.92 09.15.41 03.24.92 01.20.4203.20.92 01.16.47 03.20.92 5.19.92 2 × 20 IE 05.25.92 06.09.92 05.09.5103.18.92 01.15.14 03.20.92 05.07.44 03.30.92 5.19.92 2 × 20 IE 05.25.9206.09.92

Table 13A shows the date of the first immunization with the conjugatesand the immunization dose for each subject, as well as tuberculin (PPD)reaction and general reactions. Table 13B shows the same data for thesecond conjugate immunization. Table 13C shows the same data for thethird conjugate immunization, and Table 13D shows the same data forthose subjects immunized with the conjugates a fourth time (boosted).

TABLE 13A Tuberculin reaction, systematic reactions after Immunization#1 Date of Mantoux Immunization #1 Tuberculin reaction Birth 2 IE/ResultDate Dose Itching, Pain, Stiffness, Redness, Swelling General Reactions08.24.46 01.27.92 neg drop out drop out drop out 05.11.46 01.27.92 neg02.05.92 2 × 25 IE le: positive, not excessive none ri: positive, notexcessive 11.13.54 01.27.92 pos 0.2.10.92 2 × 25 IE le: positive, notexcessive none ri: positive, not excessive 08.12.27 01.27.92 pos02.07.92 2 × 25 IE le: positive, not excessive none ri: positive, notexcessive 10.13.50 01.27.92 neg 02.05.92 1 × 100 IE positive, notexcessive unwell Day 1, slight 02.02.33 01.27.92 neg 02.05.92 1 × 100 IEpositive, not excessive none 11.06.35 01.27.92 pos 02.07.92 2 × 25 IEle: positive, not excessive ri: positive, not excessive 10.05.6601.31.92 neg 02.07.92 2 × 25 IE le: positive, not excessive none ri:positive, not excessive 03.20.37 01.27.92 pos 02.07.92 2 × 25 IE le:positive, not excessive none ri: positive, not excessive 04.28.7001.27.92 neg 02.05.92 2 × 25 IE le: positive, not excessive none ri:positive, not excessive 10.17.51 01.27.92 pos 02.07.92 2 × 25 IE le:positive, not excessive none ri: positive, not excessive 03.28.5601.27.92 neg drop out drop out drop out 03.10.50 01.31.92 neg 02.07.92 1× 100 IE positive, not excessive none 10.09.48 01.28.92 neg 02.05.92 2 ×25 IE le: positive, not excessive none ri: positive, not excessive02.09.31 01.28.92 pos 02.07.92 2 × 25 IE le: positive, not excessivesleepy Days 1 + 2, slight ri: positive, not excessive 10.07.58 01.28.92neg 02.05.92 1 × 100 IE positive, not excessive none 12.30.28 01.28.92pos 02.07.92 2 × 25 IE le: positive, not excessive headache Days 1 + 2,slight; unwell Day 1, slight ri: positive, not excessive 04.21.3001.28.92 pos 02.07.92 2 × 25 IE le: positive, not excessive, blisteringnone ri: positive, not excessive, blistering 09.15.41 01.26.92 pos02.07.92 2 × 25 IE le: positive, not excessive, blistering headache +unwell Day 1, slight ri: positive, not excessive, blistering 01.20.4201.28.92 pos 02.07.92 2 × 25 IE le: positive, not excessive none ri:positive, not excessive 01.16.47 01.28.92 pos 02.07.92 2 × 25 IE le:positive, not excessive none ri: positive. not excessive 05.09.5101.28.92 neg 02.05.92 2 × 25 IE le: positive, not excessive none ri:positive, not excessive 01.15.44 01.28.92 pos 02.07.92 2 × 25 IE le:positive, not excessive none ri: positive, not excessive 05.07.4402.10.92 neg 02.17.92 2 × 25 IE le: positive, not excessive none ri:positive, not excessive

TABLE 13B Tuberculin reaction, systematic reactions after Immunization#2 Date of Mantoux Immunization #2 Tuberculin reaction Birth 2 IE/ResultDate Dose Itching, Pain, Stiffness, Redness, Swelling General Reactions08.24.16 01.27.92 neg drop out drop out drop out 05.11.46 01.27.92 neg02.05.92 2 × 25 IE le: positive, not excessive none ri: positive, notexcessive 11.13.54 01.27.92 pos 02.10.92 2 × 25 IE le: positive, notexcessive none ri: positive, not excessive 08.12.27 01.27.92 pos02.07.92 2 × 25 IE le: positive, not excessive none ri: positive, notexcessive none 10.13.50 01.27.92 neg 02.05.92 1 × 100 IE positive, notexcessive headache Days 1 + 2, slight 02.02.33 01.27.92 neg 02.05.92 1 ×100 IE positive, not excessive 11.06.35 01.27.92 pos 02.07.92 2 × 25 IEle: positive, not excessive none ri: positive, not excessive 10.05.6601.31.92 neg 02.07.92 2 × 25 IE le: positive, not excessive none ri:positive, not excessive 03.20.37 01.27.92 pos 02.07.92 2 × 25 IE le:positive, not excessive none ri: positive, not excessive 04.28.7001.27.92 neg 02.05.92 2 × 25 IE le: positive, not excessive none ri:positive, not excessive 10.17.51 01.27.92 pos 02.07.92 2 × 25 IE le:positive, not excessive none re: positive, not excessive 03.28.5601.27.92 neg drop out drop out drop out 03.10.50 02.31.92 neg 02.07.92 1× 100 IE positive, not excessive headache Day 2, slight 10.09.4801.28.92 neg 02.05.92 2 × 25 IE le: positive, not excessive none ri:positive, not excessive 02.09.31 01.28.92 pos 02.07.92 2 × 25 IE le:positive, not excessive fever 38.6° C. + heavy legs Day 1, slight ri:positive, not excessive 10.07.58 01.28.92 neg 02.05.92 1 × 100 IEpositive, not excessive none 12.30.28 01.28.92 pos 02.07.92 2 × 25 IEle: positive, not excessive dizzy Days 1-3; fever Day 1 38.6° C.; Day37° C. ri: positive, not excessive 04.21.30 01.28.92 pos 02.07.92 2 × 25IE le: positive, not excessive none ri: positive, not excessive 09.15.4101.28.92 pos 02.07.92 2 × 25 IE le: positive, not excessive no appetiteDays 2 + 3, slight ri: positive, not excessive 01.20.42 01.28.92 pos02.07.92 2 × 25 IE le: positive, not excessive none ri: positive, notexcessive 01.16.47 01.28.92 pos 02.07.92 2 × 25 IE le: positive, notexcessive none ri: positive, not excessive 05.09.51 01.28.92 neg02.05.92 2 × 25 IE le: positive, not excessive headache Day 1, moderateunwell + nausea Day 2, slight ri: positive, not excessive 01.15.4401.28.92 pos 02.07.92 2 × 25 IE le: positive, not excessive none ri:positive, not excessive 05.07.44 02.10.92 neg 02.17.92 2 × 25 IE le:positive, not excessive none ri: positive, not excessive

TABLE 13C Tuberculin reaction, systematic reactions after Immunization#3 Date of Mantoux Immunization #3 Tuberculin reaction Birth 2 IE/ResultDate Dose Itching, Pain, Stiffness, Redness, Swelling General Reactions08.24.46 01.27.92 neg drop out drop out drop out 05.11.46 01.27.92 neg02.05.92 2 × 25 IE le: positive not excessive none ri: positive, notexcessive 11.13.54 01.27.92 pos 02.10.92 2 × 25 IE le: positive, notexcessive none ri: positive not excessive 08.12.27 01.27.92 pos 02.07.922 × 25 IE le: positive, not excessive none ri: positive, not excessive10.13.50 01.27.92 neg 02.05.92 1 × 100 IE positive, not excessive none02.02.33 01.27.92 neg 02.05.92 1 × 100 IE positive, not excessive none11.06.35 01.27.92 pos 02.07.92 2 × 25 IE le: positive, not excessiveheadache Day 1, slight; dizzy Day 2, slight ri: positive, not excessive10.05.66 01.31.92 neg 02.07.92 2 × 25 IE le: positive, not excessivenone ri: positive, not excessive 03.20.37 01.27.92 pos 02.07.92 2 × 25IE le: positive, not excessive none ri: positive, not excessive 04.28.7001.27.92 neg 02.05.92 2 × 25 IE le: positive, not excessive none ri:positive, not excessive 10.17.51 01.27.92 pos 02.07.92 2 × 25 IE le:positive, not excessive, blister none re: positive, not excessive03.28.56 01.27.92 neg drop out drop out drop out 03.10.50 01.31.92 neg02.07.92 1 × 100 IE positive, not excessive none 10.09.48 01.28.92 neg02.05.92 2 × 25 IE le: positive, not excessive none ri: positive, notexcessive 02.09.31 01.28.92 pos 02.07.92 2 × 25 IE le: positive, notexcessive sleepy, listless Days 1 + 2 ri: positive, not excessivemoderate, Fever 37.5-38° C. 10.07.58 01.21.92 neg 02.05.92 1 × 100 IEpositive, not excessive none 12.30.28 01.28.92 pos 02.07.92 2 × 25 IEle: positive, not excessive fever 37° C. Days 0-2 ri: positive, notexcessive 41.21.30 01.28.92 pos 02.07.92 2 × 25 IE le: positive, notexcessive, but wide earbuzzing ri in evening, slight ri: positive, notexcessive 09.15.41 01.28.92 pos 02.07.92 2 × 25 IE le: positive, notexcessive, blister none ri: positive, not excessive, blister 01.20.4201.28.92 pos 02.07.92 2 × 25 IE le: positive. not excessive none ri:positive, not excessive 01.16.47 01.28.92 pos 07.02.92 2 × 25 IE le:positive, not excessive none ri: positive, not excessive 05.09.5101.28.92 neg 05.02.92 2 × 25 IE le: positive, not excessive none ri:positive, not excessive 01.15.44 01.28.92 pos 07.02.92 2 × 25 IE le:positive, not excessive none ri: positive. not excessive 05.07.4402.10.92 neg 07.02.92 2 × 25 IE le: positive, not excessive none ri:positive, not excessive

TABLE 13D Tuberculin reaction, systematic reactions after Immunization#4 Date of Mantoux Immunization #4 Tuberculin reaction Birth 2 IE/ResultDate Dose Itching, Pain, Stiffness, Redness, Swelling General Reactions08.24.46 01.27.92 neg drop out drop out drop out 05.11.46 01.27.92 neg11.13.54 01.27.92 pos 02.10.92 2 × 25 IE le: positive, not excessivenone ri: positive, not excessive 08.12.27 01.27.92 pos 02.07.92 2 × 25IE le: positive, not excessive none ri: positive, not excessive 10.13.5001.27.92 neg 02.02.33 01.27.92 neg 11.06.35 01.27.92 pos 05.19.92 2 × 25IE le: positive, not excessive headache, unwell, nausea Day 2, slightri: positive, not excessive 10.05.66 01.31.92 neg 03.20.37 01.27.92 pos02.07.92 2 × 25 IE le: positive, not excessive none ri: positive, notexcessive 04.28.70 01.27.92 neg 10.17.51 01.27.92 pos 02.07.92 2 × 25 IEle: positive, not excessive, blister none re: positive, not excessive03.28.56 01.27.92 neg drop out drop out drop out 03.10.50 01.31.92 neg10.09.48 01.28.92 neg 02.09.31 01.28.92 pos 10.07.58 01.28.92 neg12.30.28 01.28.92 pos 02.07.92 2 × 25 IE le: positive, not excessivedizzy Day 1, moderate, ri: positive, not excessive fever 37.6-38.6° C.Day 2, slight 04.21.30 01.28.92 pos 09.15.41 01.28.92 pos 01.20.4201.28.92 pos 01.16.47 01.26.92 pos 02.07.92 2 × 25 IE le: positive, notexcessive none ri: positive, not excessive 05.09.11 01.28.92 neg01.15.44 01.28.92 pos 05.07.44 02.10.92 neg 02.17.92 2 × 25 IE le:positive, not excessive none ri: positive, not excessive

Table 14 shows the total amount of anti-MN peptide antibody produced byPPD skin test negative and skin test positive subjects after conjugateimmunizations at days 1, 14, 28 and 42. The amount of antibody producedis shown by the reciprocal titers of antibody that recognize the MNpeptide. No PPD-negative subjects mounted a significant antibodyresponse. However, almost all of the PPD-positive subjects did so afterthe third primary immunization. The PPD skin test positive subjectsimmunized three times with the conjugates produced the greatest amountof antibody with GMT (geometric mean titer) rising by nearly 10-fold.GMT was derived by picking an OD value and its respective serum dilutionvalue in the linear range of the assay, and then multiplying the serumdilution value by the OD.

TABLE 14 Total anti-MN-PND antibody after immunization with theMN-PND-PPD conjugate vaccine Mean(A₄₀₅) (range) No. of Group Day 1 Day14 Day 28 Day 42 Positives/Total PPD Skin test <0.1 0.153 0.201 0.2262/9  negative (0.086-0.224) (0.129-0.306) (0.038-0.351) PPD Skin test<0.1 0.170 0.354 0.556 9/13 positive (0.106-0.238)  (0.11-0.753)(0.104-1.107)

Table 15 shows, by reciprocal titer, the total amount of anti MN-PNDantibody before and after the booster (fourth) immunization with theconjugates. Six the seven PPD skin test positive subjects responded tothe booster immunization with a significant antibody response. Patient24, who was PPD-negative and therefore not expected to respond, wasincluded as a negative control.

TABLE 15 Reciprocal Titer of anti-MN-PND antibody before and afterboosting Titer Volunteer No. Before After  3 20 100  4 20 50  7 20 >500 9 20 >500 11 20 >500 15 20 17 20 20 18 20 19 20 20 <20 21 <20 20  24*<20 <20 *PPD-negative

Table 16 shows which subjects produced high affinity/avidity antibodiesin response to conjugate immunizations as well as the amounts of highaffinity/avidity antibody produced by each subject, as determined by theantigen-limited ELISA described herein. PPD skin test positive subjectswho received the booster immunization, subjects 7, 9, 11, 15, 17, 18 and19, produced antibodies which were higher affinity/avidity. The highestaffinity antibodies were produced by subjects 17 and 18. Boosting had noeffect on the PPD-negative subject.

TABLE 16 High affinity antibody from PPD skin test positive volunteersAmount (ng/well) MN-PND, which yielded Serum Probe A₄₀₅ ± 0.2* NormalSerum >500 Positive Control 5 Volunteer No.  7 50  9 100 11 50 15 10 175 18 5 19 50 *High affinity antibodies are those reacting with a coatingconcentration of <100 ng/well yielding an absorption of >0.2.

Table 17 shows inhibitory activity to HIV infection as evidenced by areduction in the amount of P24 antigen produced after titer HIV-1 MN wasincubated with the sera of six out of seven vaccinated subjects. Thus,antibodies produced in response to the conjugate vaccines of thisinvention are able to neutralize the MN strain of HIV. See data forsubjects 7, 9, 15 and 17. Therefore, the vaccines of this invention areuseful in the treatment and transmission prevention of HIV.

TABLE 17 Neutralization activity after third immunization with vaccinespg p 24-Antigen/ml (% Reduction) Experiment Serum 1:10 (Reduction) Serum1:100 (Reduction) Negative Control 241806 242305 Volunteer No.  2 59105(76%) 157700 (36%)  7 290 (99.9%) 5356 (98%)  9 181 (99.9%) 285 (99.9%)15 35729 (85%) 23729 (90%) 17 62813 (74%) 33137 (87%) 18 308605 (0%)42581 (82%) 19 130805 (46%) 191861 (21%) These data are following 18days of culturing H9 cells infected with HIV-1 MN (approx. 100 TCID₅₀)incubated with sera of vaccinated subjects.

The above subjects which produced anti-MN peptide high affinity/avidityantibodies were vaccinated with the MN peptide (KRIHIGPGRAFYT) (SEQ IDNO:1) covalently coupled to carrier PPD. As discussed hereinabove, theconjugate vaccines of this invention may be administered alone, or in a“cocktail” of several conjugates. The conjugates of the cocktail maycomprise different peptides, which peptides may be cross-reactive withthe high affinity/avidity antibodies. For example, the MN peptide(KRIHIGPGRAFYT) (SEQ ID NO:1) may be used to immunize subjects. Theantibodies produced by the immunized subjects are cross-reactive inhumans with other peptides, such as (GPGRAF)(SEQ ID NO:16)3, IYIGPGRAC(SEQ ID NO:2), IAIGPGRAC (SEQ ID NO:3), IHIGPGRAC (SEQ ID NO:4),RSIHIGPGRAFYA (SEQ ID NO:6), KSITKGPGRVIYA (SEQ ID NO:7), KGIAIGPGRTLYA(SEQ ID NO:8) and SRVTLGPGRVWYT (SEQ ID NO:9). This is shown in Table 18below. Table 18 shows the ability of sera from volunteers immunized with3 doses of MN-PND-PPD to recognize heterologous V3 loop PND peptides. ODrepresents the optical density obtained in ELISA plates coated with aspecific PND-peptide of the V3 loop. It should be noted that vaccine No.3 demonstrated a reactivity to 3 peptides (peptide Nos. 354, 355 and358) at 1:20 serum dilution. Vaccine No. 7 demonstrated a broaderreactivity at serum dilutions 1:20 and 1:100 (to peptide Nos. 282, 285,354, 355 and 358).

TABLE 18 Cross-Reactive Antibodies In Humans DILUTION 282-(GPGRAF)3283-IYIGPGRAC 284-IAIGPGRAC 285-IHIGPGRAC 354-RSIHIGPGRAFYA CONTROL1:20  0.139 0.126 0.119 0.253 0.083  #3 1:20  0.158 0.087 0.117 0.1030.799 1:100 0.057 0.036 0.041 0.055 0.172 1:200 0.035 0.026 0.032 0.0470.119 1:500 0.026 0.022 0.024 0.028 0.159  #4 1:20  0.105 0.069 0.1230.129 0.940 1:100 0.037 0.028 0.036 0.085 0.296 1:200 0.029 0.021 0.0280.025 0.151 1:500 0.022 0.015 0.023 0.018 0.076  #7 1:20  0.861 0.2200.280 0.616 0.809 1:100 0.234 0.065 0.095 0.144 0.380 1:200 0.121 0.0990.064 0.102 0.277 1:500 0.048 0.027 0.036 0.058 0.117  #9 1:20  0.2180.141 0.470 0.425 1.574 1:100 0.085 0.055 0.211 0.116 0.748 1:200 0.0520.039 0.165 0.078 0.571 1:500 0.039 0.023 0.126 0.034 0.246 #11 1:20 0.478 0.210 0.436 0.465 1.936 1:100 0.099 0.060 0.129 0.145 0.838 1:2000.075 0.042 0.089 0.085 0.525 1:500 0.046 0.028 0.063 0.058 0.365 #171:20  0.266 0.257 0.312 0.242 2.106 1:100 0.161 0.095 0.136 0.083 1.1571:200 0.178 0.059 0.080 0.043 0.640 1:500 0.043 0.035 0.056 0.034 0.333#21 1:20  0.180 0.110 0.160 0.138 0.320 1:100 0.056 0.032 0.069 0.0660.081 1:200 0.038 0.025 0.045 0.037 0.113 1:500 0.102 0.020 0.034 0.0250.129 #24 1:20  0.199 0.134 0.208 0.168 0.493 1:100 0.048 0.088 0.0720.061 0.124 1:200 0.040 0.099 0.046 0.039 0.058 1:500 0.033 0.019 0.0350.021 0.038 DILUTION 355-KRIHIGPGRAFYT 356-KSITKGPGRVIYA357-KGIAIGPGRTLYA 358-SRVTLGPGRVWYT CONTROL 1:20  0.158 0.112 0.1050.095  #3 1:20  1.132 0.105 0.152 0.292 1:100 0.335 0.035 0.047 0.0511:200 0.182 0.028 0.027 0.037 1:500 0.083 0.017 0.018 0.028  #4 1:20 1.159 0.062 0.078 0.095 1:100 0.419 0.029 0.030 0.034 1:200 0.241 0.0230.022 0.024 1:500 0.108 0.016 0.018 0.016  #7 1:20  2.178 0.179 0.3590.613 1:100 1.368 0.084 0.106 0.218 1:200 0.918 0.043 0.054 0.103 1:5000.399 0.023 0.032 0.053  #9 1:20  2.181 0.103 0.145 0.222 1:100 1.2390.055 0.050 0.066 1:200 0.798 0.037 0.039 0.051 1:500 0.356 0.025 0.0270.029 #11 1:20  2.231 0.368 0.794 0.472 1:100 1.549 0.093 0.163 0.1111:200 0.982 0.067 0.087 0.075 1:500 0.445 0.041 0.057 0.047 #17 1:20 0.434 0.428 0.380 0.343 1:100 0.127 0.123 0.091 0.081 1:200 0.072 0.0820.066 0.069 1:500 0.058 0.057 0.040 0.043 #21 1:20  0.593 0.116 0.1730.114 1:100 0.167 0.037 0.038 0.039 1:200 0.089 0.030 0.040 0.030 1:5000.060 0.028 0.023 0.019 #24 1:20  0.108 0.175 0.155 0.155 1:100 0.0140.055 0.048 0.053 1:200 0.011 0.035 0.033 0.039 1:500 0.011 0.026 0.0220.024

TABLE 19 Percentage of inhibition on HIV-MN control after fourthimmunization Serum Dilution PBS Media 1:25 1:100 1:250 HIV-MN Control48,510 50,360 46,380 Control Serum 22,800 20,190 24,360 1:100 3/500063,690 (31.8%) 69,700 (38.4%) 69,440 (49.7%) 1:200 4/50 10,348 (78.7%)25,900 (48.6%) 27,120 (41.6%) >1:500 7/50 896 (98.2%) — 79,860 (72.1%)+282 >1:500 9/50 7,099 (85.4%) 16,020 (68.2%) 34,810 (25.05%)+282 >1:500 11/50 1,808 (96.3%) 8,137 (83.9%) 8.708 (82.3%) +282 >1:50017/50 0.2 (99.0006%) — — −354 1:100 21/5000 15,560 (68.0%) 16,530(67.2%) 58,760 (26.6%) 1:20 24/0 52,000 (7.1%) 20,890 (58.6%) 19,740(57.5%) Percentage of inhibition on HIV-MN control

The subjects were immunized a fourth time with the PPD-MN-PPDconjugates. Table 19 shows the percentage of inhibition on HIV-MNcontrol.

Table 20 shows the affinity of antibody before and after the booster(fourth) immunization with the conjugates. As shown in Table 20, theaffinity after boosting increased dramatically.

TABLE 20 Affinity of antibody before and after boosting MN-PND (μg/ml)Volunteer No. Before* After  3 >5000 5000  4   1000 50  7 500/1000 50  9500/1000 50 11 500/1000 50 15 100/500  17 50/500 5 18 50/500 19 500/500 20 >5000 21 >5000 5000  24** >5000/>5000  >5000 *2 determinations**PPD-negative

EXAMPLE X

Immunization of Humans with Conjugates of MN-PND Coupled to Carrier PPD

Subjects who were negative in a third generation HIV antibody ELISAassay were further screened for tuberculin (PPD) reactivity with 100 mlof 2 TU/PPD administered intradermally. For the 9 subjects who werenegative to borderline PPD positive, a second skin test was performedwith 10 TU/PPD. A total of 12 subjects were classified as PPD-positiveat the 2 TU test dose.

Again, the vaccine administered was a conjugate of PPD and the MN-PNDpeptide. The MN-PND peptide sequence, KRIHIGPGRAFYT (SEQ ID NO:1), wasprepared by standard solid phase synthesis and its composition wasverified on an automated amino acid analyzer. The MN-PND peptide wasconjugated to PPD in a Pilot GMP Laboratory by reacting PPD and MN-PNDin a 2% glutaraldehyde solution (Fluka Chemicals, Buchs, Switzerland).After dialysis against sterile phosphate buffered saline, the solutionwas aseptically aliquoted at approximately 830 mg conjugate/ml. Giventhe heterogeneous nature of PPD, it was not possible to accuratelydetermine the ratio of PPD to MN-PND in the conjugate by amino acidanalysis. Therefore, incorporation of 125I MN-PND peptide into PPD inthe presence of glutaraldehyde was determined for a representative pilotlot of 600,000 cpm/mg of 125I MN-PND.

Following conjugation with 2 mg of PPD (100 TU), 710,000 cpm weredetected, which, assuming total recovery, corresponded to 0.645 mgMN-PND/mg of PPD. A human vaccine dose of 50 TU of PPD thereforecontained approximately 0.645 mg of MN-PND. The molecular weight of theconjugate was approximately 15,000 kDa as determined by HPLC. Sterilityand general safety tests were performed according to guidelines setforth by the European Pharmacopoeia. All lots of vaccine were found tobe sterile and nontoxic.

In order to immunize, the following procedure was followed: a singledose, equal to 50 TU (1 mg) of PPD, contained approximately 0.645 mg ofthe MN-PND peptide in a volume of 100 ml. Subjects were immunized with50-100 TU of PPD conjugate vaccine (0.645-1.29 mg of MN-PND) by theintradermal route into one or both inner forearms. A pilot experimentexamining the immunogenicity of the MN-PND-PPD conjugate by immunizingfive PPD positive volunteers with 2, 10, or 50 TU of vaccine conjugateon days 0, 60 and 90 indicated that antibody responses were only notedin the volunteers receiving 50 TU. In addition, pilot experimentsindicated that 100 TU of MN-PND-PPD conjugate could be safelyadministered to PPD-negative subjects. Based on these results, 24additional volunteers were immunized with either 50 TU for PPD positivevolunteers of 100 TU for PPD negative individuals of the PPD conjugatevaccine on days 0, 14, 28, 82 and 383 (see Table 21, below). Allvaccinees were monitored periodically for local and systemic reactionsas well as by a standard battery of blood chemistries and complete bloodcounts. Local reactions were monitored within 1 hour, 24 hours and 72hours of immunization. In cases of local itching, redness and swellingno topical treatment was administered. However, at the first sign ofblistering, topical corticosteroids were prescribed.

TABLE 21 Immunization Schedule PPD skin test Immunization Immunization #day Reactivity Intradermal dose I II III IV V adverse reaction Vaccinee2TU 10TU # sites days (at immunization #)  1 − − nd nd nd nd nd nd dropout  2 − − 2 × 25TU 0 14 28 nd nd none  3 + nd 2 × 25TU 0 14 28 82 ndnone  4 + nd 2 × 25TU 0 14 28 82 383 none  5 − − 1 × 100TU 0 14 28 nd ndnone  6 − − 1 × 100TU 0 14 28 nd 383 none  7 + nd 2 × 25TU 0 14 28 82383 none  8 − + 2 × 25TU 0 14 28 nd nd none  9 + nd 2 × 25TU 0 14 28 82nd none 10 − − 2 × 25TU 0 14 28 nd nd none 11 + nd 2 × 25TU 0 14 28 82383 blister (IV; V) 12 − nd nd nd nd nd nd nd drop out 13 − − 1 × 100TU0 14 28 nd 383 none 14 − − 2 × 25TU 0 14 28 nd 383 none 15 + nd 2 × 25TU0 14 28 82 382 low grade fever 16 − − 1 × 100TU 0 14 28 nd 385 none 17 +nd 2 × 25TU 0 14 28 82 383 low grade fever 18 + nd 2 × 25TU 0 14 28 82nd blister (I) 19 + nd 2 × 25TU 0 14 28 82 nd blister (I; III) 20 + nd 2× 25TU 0 14 28 82 nd none 21 + nd 2 × 25TU 0 14 28 82 nd none 22 − + 2 ×25TU 0 14 28 nd nd none 23 − nd 2 × 25TU 0 14 28 nd nd none 24 − − 2 ×25TU 0 14 28 82 nd none

Since preclinical studies have shown in guinea pigs that the PPD-MN-PNDconjugate has induced strong antibody responses only in BCG primed, PPDskin test positive animals, the immune response in volunteers wasstratified to those who were PPD positive and those who were PPDnegative. After 3 immunizations, vaccine boosters were given exclusivelyto PPD skin test positive volunteers (see Table 21).

The presence of antibodies directed against MN-PND was measured asdescribed above. 100 mg/well of a solution of MN-PND (5,000 ng/ml) in100 mM NaHCO₃, pH 9.6, was incubated in 96-well flat-bottomed microtiterELISA plates overnight (MN-PND titer plates). Following peptide coating,the plates were washed with PBS containing 0.05% Tween-20, and unboundsites were blocked with blocking buffer (PBS/0.55 casein/0.05%Tween-20/0.001% rhodamine). Serial dilutions of the sera to be testedwere added to the wells at a 1:20 dilution in blocking buffer andincubated for 1 hour at 37° C. Unbound antibodies were washed away andthen goat anti-human IgG conjugated to peroxidase was added, incubatedfor 1 hour at 37° C. and then washed. Substrate was added and absorbanceat 405 nm was measured with an automated spectrophotometer (TitertekMultiscan, Flow Laboratories, McLean, Va.). Samples that gave an opticaldensity (OD) value greater twice the background absorbance wereconsidered seroreactive. The data is presented as the reciprocal titerof the highest serum dilution that was seroreactive.

High affinity antibodies were measured by an antigen-limited ELISA ashas been described. Microtiter plates were coated overnight with 5000,1000, 500, 100, 50, 10, 5, or 0 ng/ml of MN-PND (MN-PND affinityplates). Following peptide coating, the plates were processed asdescribed above. The anti-MN-PND affinity was measured by incubating a1:20 dilution of serum in the MN-PND titer plates and reactivity wasdetermined as described above. Therefore, reactivity with lower antigenconcentrations correlated with higher affinity antibody, and theaffinity is reported as the lowest antigen concentration wherereactivity was detected. The samples were coded and run with positiveand negative controls.

It was determined that there was cross-reactivity of the generatedantibody response, in addition to the classical MN-PND peptide used inthe vaccine (KRIHIGPGRAFYT) (SEQ ID NO:1), 8 additional peptidesrepresenting different HIV-1 strains were synthesized. ELISA plates werecoated with an optimal concentration of the peptide, as described above.The peptides used were: IYIGPGRAC (SEQ ID NO:2), IAIGPGRAC (SEQ IDNO:3), IHIGPGRAC (SEQ ID NO:4), a triple repeat of the V₃ loop capsequence-GPGRAFGPGRAFGPGRAF (SEQ ID NO:5), the PND sequence ofHIV-1_(SC)-RSIHIGPGRAFYA (SEQ ID NO:6), the PND sequence ofHIV-1_(RF)-KSITKGPGRVIYA (SEQ ID NO:7), the PND sequence ofHIV-1_(NY-5)-KGIAIGPGRTLYA (SEQ ID NO:8) and the PND sequence ofHIV-1_(CDC-42)-SRVTLGPGRVYWYT (SEQ ID NO:9), Seroreactivity wasdetermined as described above.

In order to determine whether salivary and serum antibodies wereproduced, total saliva was collected from two HIV-1 negative volunteers,from HIV positive volunteers, from HIV positive volunteers and from 4vaccinees, utilizing the Orasure hypertonic sponge system (Epitope,Beaverton, Oreg., USA). This system has been used in the past for thedetection of HIV-1 specific salivary IgA. Since the preservative in theOrasure vial appeared to partially denature the IgA, it was replaced bybuffered saline (PBS) with 0.1 M sodium azide. Saliva was diluted 1:20in blocking solution, and 1 ml were added to each well of MN-PND titerplates. After 1 hour of incubation the plates were washed with PBScontaining 0.05% Tween-20, reacted with peroxidase conjugated anti-humanIgA or to anti-human secretory component for one hour, washed, incubatedwith substrate and absorbance was measured as described above.

In order to perform neutralization assays the following procedure wasfollowed: HIV-1_(MN) strain obtained from the AIDS Reference and ReagentProgram, passaged in H9 cells and the TCID₅₀ was determined asdescribed. H9 HIV-1_(MN) cells chronically infected with HIV-1_(MN) werewashed and passed into fresh RPMI containing FCS for 24 hours. Infectedcells were separated from free viral particles by centrifugation at1,000 RPM for 30 minutes at 4° C. The cell-free supernatant wasaliquoted and stored at −70° C. The infectious titer of the virus wasdetermined by incubation of serial one-to-ten dilutions of viralsupernatant in 5 ml of growth media containing H9 cells (10⁶/ml). Afterculturing the cells for 5 days, reverse transcriptase activity of thesupernatant was determined. The highest dilution of the virus stock thatinfected half of the quadruplicate cultures of H9 cells as determined byassessment of the supernatant for p24 antigen content using a commercialkit (DuPont, Wilmington, Del.) was defined as on TCID₅₀. Followingheat-inactivation, the serum to be tested for neutralizing activity wasincubated at the indicated dilution with 10² to 10³ TCID₅₀ of HIV-1_(MN)for 1 hour at 37° C. and then cultured with H9 cells. Following 18 daysof culture, the concentration of P24 antigen in an aliquot ofsupernatant was measured using an antigen capture assay (DuPont,Wilmington, Del.). In addition, the presence of syncytia was sorted bymicroscopic assessment.

The proliferative response of PBMC from vaccinated volunteers wasassessed. Peripheral blood mononuclear cells (PBMC) were separated onFicoll gradients, resuspended in RPMI culture medium with 10% human ABserum and 10⁵ cells in 100 ml culture medium were placed in each well of96 well flat-bottomed microtiter plates and incubated with and withoutthe MN-PND peptide (1 mg/ml). After 5 days of culture, proliferativeresponses were assessed by determining the incorporation of ³H-thymidineadded during the last 16 hours of culture.

The presence of MN-PND-specific CTL was then determined. Principally,EBV-transformed autologous B cells were incubated with vaccinia vectors(VPE 16-HIV-1 gp160-IIIB and v PEMS-HIV-1/gp160-MN) for 90 minutes at37° C. Cells were then washed and transferred to a 24-well culture platefor overnight incubation at 37° C. Subsequently, target cells werelabelled with ⁵¹Cr and after three washes were resuspended in RPMI with10% FCS at 5×10⁴ viable cells per well. The subjects' effector PBMC weresuspended at 2×10⁶ viable cells per ml and reacted with magneticmicrospheres (Dynabeads, Advanced Magnetics, Boston, Mass.) coated withmonoclonal antibodies to CD4. After 45 minutes incubation at 5° C. thetube was placed on the capture magnet. The cell suspension not bound bythe magnet was resuspended to 5×10⁶ cells per ml in RPMI+10%/FCS. 100 mlof each effector cell population was added to triplicate wells of thetarget cells and ⁵¹CR release was measured. Spontaneous release controlsas well as maximal release with 0.5% Triton X-100 were runsimultaneously.

Due to the development of adverse responses to the vaccine, twosubjects, #1 and #12, dropped out of the study prior to receiving thefirst vaccine dose. Reactions following each dose of vaccine aredetailed in Table 21. Typical reactions were local redness andinduration after 24-48 hours. Severe local reactions were not observed.Small painless blisters which resolved without scarring occurred invaccine subjects #11, #18 and #19. In vaccine subject #18 the blisterappeared only in the first of 4 immunizations, and in vaccine subject#11 a blister was noted only after the 3rd but not after the 4thimmunization. In the other vaccine subjects, the skin reactions did notincrease with subsequent doses. Two vaccine subjects, #15 and #17,experienced low grade fever one day after an immunization but not afteranother boost.

Antibody responses to MN-PND were determined. In a pilot study usingfive PPD+ subjects, only one subject receiving the highest vaccine dose(50 TU=0.65 mg PND) had an antibody response which increased after thesecond and third dose. These antibodies were of high affinity. The samesubject's PBMC responded in vitro by proliferation and secretion. ofIL-2 upon exposure to MN-PND. Subsequently all PPD+ subjects receivedthe 50 TU PPD-MN-PND dose (0.65 mg PND) whereas some of the PPD negativesubjects received up to 100 TU-PPD-MN-PND. None of the PPD negativesubjects had any antibody response to the PND. In all 12 PPD positivevaccine subjects reciprocal antibody titers ranging between 1:100 and1:2,000 were detected. The majority of vaccine subjects had a titer of1/500 or above (see Table 22, below). Although vaccine subject #23 hadthe lowest antibody titer, these antibodies were of high affinity (seeTable 22).

TABLE 22 Reciprocal antibody titer, and affinity to MN-PND Days AfterPrimary Immunization VACCINEE # 0 14* 28* 42 82 98 131 169 179 251 383392 449  3 —+ <1/20+ >1/20+ 1/20 1/20 1/100 1/50 (100) 1/50 (5000)+ (5)(100) (100)  4 —+ <1/20+ —+ 1/20 1/20 1/200 1/50 1/50 (500) (500)+ (50)(100)+ (100)  7 —+ <1/20+ >1/20+ 1/200 1/500 1/1000 1/500 1/200 1/20(250) (500)+ (10) (5)+ (50) (100)  9 —+ 1/20+ >1/20+ 1/200 1/20 1/1000(250) (500)+ (50) 11 —+ <1/20+ >1/20+ 1/200 1/200+ 1/1000 >1/50 nd (100)(10) (10)+ (100) 15 —+ <1/20+ >1/20+ 1/200 1/100 1/500 1/500 1/200 1/200(100) (500)+ (10) (10)+ (10) (5) 17 —+ <1/20+ >1/20+ 1/200 1/200 1/20001/500 1/500 1/500 (50) (500)+ 5 (10)+ (10) (10) 18 —+ <1/20+ >1/20+1/200 1/200 1/1000 1/500 (50) (500)+ (5) (5) 19 —+ <1/20+ >1/20+ 1/2001/100 (100) (500)+ 20 —+ <1/20+ nd+ nd 1/100 <1/20 1/20 (5000)+ (5000)(5000) 21 —+ <1/20+ nd+ <1/20 1/20 >1/20 >1/100 (5000)+ 0 (50) (50) 23—+ <1/20+ <1/20+ <1/20 nd+ 1/100 1/100 (50) (50) * — only 1/20 serumdilution was used + — immunization nd — not done ( ) — affinity ngPND/well

Next, the affinity of anti-PND serum antibodies was determined. Anyreactivity with wells coated with 100 mg/ml or less of MN-PND in theELISA plates was considered as high affinity. No high affinityantibodies could be detected in any vaccinee until after the third boost(see Table 22 and FIG. 4). High affinity antibodies persisted thereafterfor up to 367 days (see Table 22 and Table 23, below). There was afurther increase in affinity/avidity to the highest measurable level insubjects #7, #15 and #17. The time course of the antibody titers andaffinities for a representative volunteers, #17, is presented in FIG. 4.

TABLE 23 Persistance of Antibody Titer and High Affinity Antibodies Tothe MN-PND, Days After 2nd, 3rd and 4th Boost DAYS AFTER BOOST Vaccinee# boost 2 boost 3 boost 4 3 >54* 367 >66 4 >54* 301 >66 7 >54* 301 >669 >54* >49 not done 11 >54* 301 not done 15 >54* 301 >66 17 >54* 301 >6618 >54* >169 not done 19 >54* not done not done 20 none >169 not done 21none >169 not done 23 none >223 not done *low affinity antibodies

Cross-reactivity of serum antibodies was determined. It was found thatthere was strong recognition of peptides from HIV-1_(SC) (FIG. 5) whichshares the internal IHIGPGRAFY (SEQ ID NO:15) sequence with the MNstrains. Reactivity against other peptides such as RF:NY-5,CDC-42 wasalso detectable. The cross-reactivity did increase after the fourthboost (not shown). PPD-negative volunteer ™21 exhibited borderlinereactivity with MN with some cross-reactivity to SC.

Virus neutralization was then determined. None of the sera from PPDnegative volunteers neutralized HIV-1_(MN) (not shown). The ability ofsera from PPD positive immunized subjects (after the 3rd boost) toneutralize the MN strain of HIV-1 is shown in FIG. 6. Sera from 10 of 11subjects showed neutralizing activity at dilutions of up to 1:200(subjects #4, #9 and #11) and at 1:100 (subjects #4, #7, #9, #11, #15,#17, #18 and #21). In two subjects (#7 and #9) there was a >99%reduction of p24 in culture supernatants with sera diluted 1:100.

Next, lymphocyte proliferative responses to MN-PND were determined. Theproliferative responses to the MN-PND peptide were tested in 4 subjects(#18, #20, #21 and #23). Subject #23 exhibited a good in vitroproliferative response to the peptide after the 3rd boost (see FIG. 7).

Serum and salivary IgA were then determined. All PPD positive vaccinesubjects who had an IgG response to the MN-PND also had specific IgAantibodies to MN-PND present in their serum. In some, serum IgA antibodytiters were identical to serum IgG titers (see Table 24, below). Inothers the serum IgA titers were 1/2-1/25 of serum IgG titers. Volunteer#14, who did not mount a serum IgG response, had detectable serum IgAand low titer salivary IgA to the MN-PND. In 3 out of 4 testedunconcentrated saliva specimens, specific IgA antibodies to MN-PND weredetectable at titers of up to 1/100 utilizing monoclonal antibodies tohuman IgA. In ELISAs utilizing a monoclonal antibody to bound SC no IgAwas noted in unconcentrated saliva.

TABLE 24 Serum and Salivary IgA Antibodies to MN-PND Serum Saliva DaysIgG IgA IgA after highest highest highest vaccinee # boost # boost ODtiter OD titer OD tMwer  3 3rd 49 1.13 1/100 0.59 1/100  4 3rd 55 1.161/200 0.75 1/200 4th 66 0.7 1/50  0.72 1/50  0.16 —  7 3rd 49 >2.0 1/1000 1.42 1/200 301  1.43 1/500 0.81 1/100 4th 66 1.41 1/100 1.051/100 0.33 1/50  9 3rd 49 >2.0  1/1000 1.2 1/500 11 3rd 49 1.94  1/10000.71 1/100 14 4th 66 0.06 — 0.69 1/50  0.27 1/20 15 3rd 301  1.1 1/5000.31 1/50  17 3rd 55 >2.0  1/1000 0.71 1/100 301  1.48 1/500 0.3 1/20 4th 66 0.95 1/100 0.55 1/20  0.69  1/100 18 3rd 97 1.22  1/1000 1.481/100

In studying CTL responses, three out of five vaccine subjects expressedspecific lysis. The extent of lysis was variable (see FIG. 8). In theCTL responsive vaccine subjects, both CD8⁺ and CD4⁺ MN-PND peptidespecific CTLs were noted. Vaccine subject #6 demonstrated primarily CD8responses, while vaccine subjects #7 and #15 exhibited both CD4 and CD8CTL responses.

Most immunogens that induce excellent cell-mediated immunity (CMI) aswell as systemic and mucosal humoral immune responses are liveattenuated vaccines. In fact, live attenuated simian immunodeficiencyvirus (SIV) protected rhesus monkeys from a challenge by wild-typepathogenic SIV. Live attenuated HIV vaccines are, however, unacceptablein humans for fear of reversion to virulence and induction of pathologicprocesses. The inventors have determined that an HIV-1 subunit (peptide)vaccine has induced in human subjects a broad range of immune responsesnearing those of live-attenuated vaccines.

In order to develop a meaningful strategy for a preventive subunit AIDSvaccine there is a need for precise definition of the elements ofprotective immunity. In acute HIV-1 infection and in HIV-l exposed butuninfected individuals, cytotoxic T lymphocytes (CTL) represent thefirst immune response elicited followed by antibodies to the HIVenvelope. Other studies have also indicated that immune responses to theHIV envelope, and especially anti-V₃ loop primary neutralizing domain(PND) targeted antibodies, are pivotal for protection. Recombinant HIVenvelope subunit vaccines have, therefore, received the greatestattention but have not been shown to induce potent and lasting immunity.

The vaccines of the invention produced persistent lymphoproliferativeresponses to a variety of HIV antigens preceding the induction ofneutralizing antibodies. Consistent and broad immune responses wereachieved only with the high dose regimen (300 mg/dose) and after the 3rdimmunization with the HIV-1_(IIIB) rgp120 vaccine in alum adjuvant. Thesafety of high dose vaccines has, however, been questioned for fear ofactivation of virus replication and induction of non-protectiveimmunity. It has, for example, been demonstrated that a largeintrarectal innoculum of SIV to macaques resulted in infection andantibody production with minimal CMI, whereas administration of lowerdoses elicited strong CMI with no antibody production and no detectableinfection. In addition Bretscher et al. have shown that mice injectedwith a high dose of inactivated HIV exhibited a transient CMI responsefollowed by antibody response, whereas lower doses generated apersistent CMI response. The inventors have shown that minute doses ofPPD-MN-PND administered intradermally in the absence of adjuvants caninduce protective immune responses. The vaccines of the invention, at adose of 0.65 mg, induced a consistent, potent, and long lasting humoraland cellular immune response in PPD DTHR positive HIV-1 uninfectedvolunteers.

The unique potency of this vaccine may be attributed to both theintradermal route of immunization and to the use of PPD as a carrier.PPD is a unique immunologic reagent in that virtually everyone in theworld with a functional immune response who has been exposed to BCG orM. tuberculosis infection will give a T-cell mediated DTHR to minuteamounts of PPD. One explanation for its universal T-cell recognition isthat PPD is made from cultures of autolyzed bacteria and may contain amixture of degraded an “preprocessed” antigens that can be presented byall MHC Class II hapotypes. Studies indicate that mice presensitized byBCG can produce high levels of antibodies to peptides or even tocarbohydrate epitopes if they are conjugated to PPD. PPD itself isnonimmunogenic, thereby avoiding the potential of carrier epitopicsuppression. PPD is also widely used as a human diagnostic reagent withan extensive safety data base with hardly any adverse effects except forlocal reactions that resolve without scarring spontaneously or aftertopical corticosteroids. Worldwide, billions of people are PPD DTHRpositive.

Immunization with BCG is also widely practiced in developing areas ofthe world where AIDS is prevalent, giving rise to a multi-billion primedpopulation. Because of the reemergence of tuberculosis and more recentlyof multiple drug-resistant tuberculosis, consideration is now beinggiven in Europe and the U.S. to BCG vaccinate clinically-stable HIVinfected patients and their household contacts. It is thereforebeneficial to administer BCG to prospective vaccine subjects who are PPDDHTR negative.

The antibody responses encountered in the PPD-MN-PND vaccine subjectswere uniform and at higher titers than in any previously reportedvaccine. Also of importance is the observation that the immune responseachieved was broad. Infected individuals usually have a swarm of HIV-1variants, and mutation rates are estimated at up to 1% per year.Restricted and narrow spectrum antibody response may therefore promotethe emergence of neutralization resistant strans. Such mutations havebeen observed also in the V₃ region. Fortunately, a few sequence motifsof the V₃ account for over 50% of all isolates analyzed. The variationin the PND is therefore not insurmountable in the presence of highlycross-reactive, high affinity antibodies.

The antibodies generated by a low dose of the PND vaccine of theinvention were of the highest affinity, were cross-reactive, wereHIV-1_(MN) neutralizing and were syncytium inhibiting. Sera fromimmunized subjects recognized PND peptides from the homologous MN andfrom other V₃ PND peptides. There was extensive recognition of peptidesfrom HIV-1 SC and to a lesser extent of RF; NY5 and CDC-42. Theseantibody responses exceeded those of the recombinant gp120 vaccine withwhich only the high dose (300 mg) elicited neutralizing andcross-reactive antibodies. Another advantage of the peptide vaccines ofthe invention is that additional peptides can be conjugated to PPD,rapidly, inexpensively and according to the emergence of new PNDvariants in certain geographic areas.

Ideally, a vaccine should induce both salutary antibody responses andcell-mediated immunity. Cell mediated immunity is usually broader andmay abrogate more easily the problem of mutations. To date, all subunitimmunogens tested in animals and in humans induced antibodies thatlasted at best only for several months after boosting, CMI induced wasfeeble, and dose response was less pronounced for the generation ofmemory T cells as expressed by in vitro assays of antigen specificlymphocyte proliferation. (The latter response was present early and wassustained for at least 4 months only after the third vaccine injection.)In contrast, with the PPD-MN-PPD vaccine of the invention not only dohigh-affinity, neutralizing antibodies persist for well over 1 year, butalso long lasting CMI is induced as expressed by generation of antigenspecific lymphoproliferation and induction of specific CD8⁺ and CD4⁺cytotoxic T lymphocytes (CTL).

Protective HIV vaccines should also induce mucosal immunity in thegenital tract in order to intercept the infectious virus at the mostfrequent route of transmission. Although systemic immunizationstrategies have protected macaques from intravenous challenge with SIV,they have not prevented vaginal transmission. Administration-ofinactivated whole SIV or synthetic peptides by the mucosal route alsodid not induce an effective immune response. There have been no reportsof HIV-1 candidate vaccines which induce mucosal immunity in humans.Prior to immunization with an HIV-1 gp160 subunit or any otherparenteral vaccine has been inadequate to stimulate mucosal immunity. Incontrast, the PPD-PND vaccine of the invention has induced high titerspecific anti-PND serum IgA and mucosal IgA antibodies.

EXAMPLE XI

Immunization of Guinea Pigs with Conjugates of MN-PND Coupled to theCarriers PPD Alone, Toxin A Alone, PPD with Toxin A, and PPD With BothToxin A and the Adjuvant Al(OH)₃

In order to produce the toxin A-PND vaccine, a total of 5.8 ml (30.9 mg)of toxin A-ADH was added to a sterile 10 ml vial containing a magneticstirrer. The pH was adjusted to 5.6 by the addition of 188 ml of HClusing an automated titrater. To this solution 62 mg of solid EDEC wasadded over a 6 minute period of time (dropwise). The pH dropped to 4.98.The reaction was allowed to proceed at ambient temperature for 1 hourwith gentle stirring and the pH was maintained at 4.95-5.1. Anadditional 31 mg of solid EDEC were added and the reaction was allowedto proceed for an additional 2.5 hours. The pH rapidly stabilized and pHtitration was not required. At the end of this period, the pH was 4.975.The solution was aseptically withdrawn into a sterile syringe andapplied onto a 5×56 cm Sephadex G-75 column equilibrated in sterile PBS,pH 7.4. The elution profile was monitored at 206 and 276 nm and 8.1 mlfractions collected. Fractions 35-57 (void volume fractions with a highabsorbance) were collected and pooled. The pool was found to contain 155mg protein per ml by the Lowry assay.

Next, guinea pigs, some of which were previously primed with BCG andsome of which were not, were immunized on days 0 and 14 with (a) thepeptide MN (KRIHIGPGRAFYT) (SEQ ID NO:1) conjugated with the carrier PPDalone (Groups 1-3 and 5); (b) the MN peptide conjugated with the carriertoxin A either alone or in conjunction with the carrier PPD (Groups 6-9and 14); (c) the MN peptide conjugated with both carriers PPD and toxinA, along with the adjuvant Al(OH)₃ (Groups 12 and 13); (d) the MNpeptide unconjugated with PPD (Group 4); and (e) the MN peptideunconjugated with toxin A and PPD (Groups 10 and 11). The results aresummarized in Table 25 below. Vaccines containing the peptide coupled totoxin A (Groups 6, 8, 9, 11 and 13) had significantly higher immuneresponses than the peptide coupled to PPD (Groups 1, 2, 3 and 5). Immuneresponse was further increased by adding PPD to the peptide-toxin Aconjugate (Group 11). In addition, adsorption of the adjuvant Al(OH)₃ tothe peptide-toxin A-PPD conjugate further increased immune response(Groups 12 and 13). See Table 25. Hence, the vaccines of this inventionare able to elicit an immune response to MN HIV, and are thereforeuseful in the treatment and transmission prevention of HIV.

TABLE 25 Anti-PND Antibody Response in Guinea Pigs Group Vaccine PrimingGKT (Range) 1 PPD-PND (Lot 1) Yes 8.3 (3.9-35.4) (1 mg) No 2.1 (1.7-2.8)2 PPD-PND (Lot 2) Yes 1.65 (0.9-2.8) (1 mg) 3 PPD-PND (Lot 3) Yes 1.49(1-4.7) (1 mg) 4 PPD (50 IU) + PND Yes 1.44 (0.9-2.4) (1 mg);unconjugated 5 PPD-PND (Lot 1) Yes 2.38 (0.08-5.2) (1 mg) 6 PPD-PND (1mg) + yes 201 (15.8-1300) Toxin A-PND (10 mg) 7 Toxin A-PND (1 mg) Yes2.41 (0.8-7.9) 8 Toxin A-PND (10 mg) Yes 53.3 (8.3-393) 9 Toxin A-PND(10 mg) No 33.2 (5-333) 10 Toxin A-PND (1 mg) + Yes 3 (1.7-9.3) PPD (50IU); unconjugated 11 Toxin A-PND (10 mg) + Yes 196 (55-1344) PPD (50IU): unconjugated 12 Toxin A-PND (1 mg) + Yes 105 (18-1018) PPD (50)IU + Al(OH)₃ 13 Toxin A-PND (10 mg) + Yes 912 (395-1374) PPD (0 IU) +Al(OH)₃ 14 Toxin A-PND (1 mg) + Yes 17 (0.4-153) PPD-PND (50 IU; 1 mg)

Table 26, below, shows reciprocal titers and affinity of antibodies inguinea pigs immunized with various vaccines of this invention. Asdemonstrated, some of the guinea pigs immunized with the vaccines ofthis invention produced high affinity antibodies to MN-HIV.

TABLE 26 Reciprocal titers and affinity of antibody in sera of guineapigs After Boost- After Boost- BCG(-14d) PND(MN)-PPD PND(MN)-TA(ADH)PND(MN) PPD GP # Day 28 Day 56 Day 28 Day 56 ″ 1 ug (Lot #1) 8428 1001,000 100  ″ ″ 8429 20 12,000 50 ″ ″ 8430 20 4,000 100  ″ ″ 8431 2012,000 50 ″ ″ 8432 20 8,000 50 ″ ″ 8433 20 — — ″ 8434 0 0 ″ 8435 0 0 ″8436 0 0 ″ 8437 0 20 ″ 8438 0 0 ″ 8439 0 20 ″ 1 ug (Lot #2) 8440 0 20 ″″ 8441 0 20 ″ ″ 8442 0 0 ″ ″ 8443 0 0 ″ ″ 8444 0 >20 ″ ″ 8445 0 >20 ″ 1ug (Lot #3) 8446 0 >20 ″ ″ 8447 0 >20 ″ ″ 8448 0 >20 ″ ″ 8449 0 >20 ″ ″8450 20 >20 ″ ″ 8451 0 20 ″ 10 ug 1 ug 8452 0 0 ″ ″ ″ 8453 0 20 ″ ″ ″8454 0 0 ″ ″ ″ 8455 0 0 ″ ″ ″ 8456 0 20 ″ ″ ″ 8457 0 0 ″ 1 ug (Lot #1)8458 0 — ″ ″ 8459 0 — ″ ″ 8460 0 — ″ ″ 8461 20 — ″ ″ 8462 0 — ″ ″ 8463 0— ″ 1 ug (Lot #1) 10 ug 8464 >20 — ″ ″ ″ 8465 2,000 — 500 ″ ″ ″ 8466 500— ″ ″ ″ 8467 500 — 500 ″ ″ ″ 8468 4,000 — 50 ″ ″ ″ 8469 500 — RECIPROCALTITER AFFINITY (ng/ml) After Boost- After Boost- BCG(-14d) PND(MN)-PPDPND(MN)-TA(ADH) PND(MN) PPD GP # Day 28 Day 42 Day 28 Day 42 ″  1 ug8475 0 >20 ″ ″ 8476 0 >20 ″ ″ 8477 0 >20 ″ ″ 8478 20 >20 ″ ″ 8479 0 20 ″″ 8480 0 >20 ″ 10 ug 8481 2,000 4,000 500  50 ″ ″ 8482 20 1,000  50 ″ ″8483 500 4,000  50 ″ ″ 8484 1,000 1,000  50  50 ″ ″ 8485 >20 500  50 ″ ″8486 >20 4,000  50 ″ 8487 >20 500 500 ″ 8488 >20 500 500 ″ 8489 >20 500500 ″ 8490 2,000 2,000 500  50 ″ 8491 20 1,000  50 ″ 8492 500 500 500100 ″  1 ug 1 ug 8493 20 2,000 100 ″ ″ ″ 8494 0 20 5,000   ″ ″ ″ 9495 2020 1,000   ″ ″ ″ 8496 0 20 1,000   ″ ″ ″ 8497 0 1,000 100 ″ ″ ″ 8498 0500 500 ″ 10 ug ″ 8499 1,000 1,000 500 100 ″ ″ ″ 8500 2,000 4,000  50 50 ″ ″ ″ 8501 500 2,000 100 ″ ″ ″ 8502 4,000 12,000  50  50 ″ ″ ″ 8503500 4,000  50 ″ ″ ″ 8504 500 4,000 500  50 ″  1 ug 1 ug 8505 20 500 500(Alum) ″ ″ 8506 1,000 2,000 500 500 ″ ″ 1 ug 8507 100 500 100 (Alum) ″ ″1 ug 8508 4,000 4,000 500  50 (Alum) ″ ″ 1 ug 8509 4,000 50  50 100(Alum) ″ ″ 1 ug 8510 100 500 100 (Alum) ″ 10 ug 1 ug 8511 4,000 500 500 50 (Alum) ″ ″ 1 ug 8512 1,000 1,000 500 100 (Alum) ″ ″ 1 ug 8513 4,0004,000 500 100 (Alum) ″ ″ 1 ug 8514 2,000 4,000 500  50 (Alum) ″ ″ 1 ug8515 500 8,000 500  50 (Alum) ″ ″ 1 ug 8516 4,000 4,000  50  50 ″ 1 ug(Lot #1)  1 ug 8517 500 4,000 500  50 ″ ″ ″ 8518 20 500 500 ″ ″ ″ 8519500 4,000 500 100 ″ ″ ″ 8520 20 4,000 500 ″ ″ ″ 8521 20 2,000 100 ″ ″ ″8522 20 4,000  50

EXAMPLE XII

Immunization of Rabbits with Conjugates of Various Peptides Coupled toKLH

Rabbits were immunized with vaccines comprising various peptides coupledto the carrier KLH as described above. The peptides coupled to KLH were(GPGRAF)₃ (SEQ ID NO:16)C (282), IYIGPGRAC (SEQ ID NO:2)(283), IAIGPGRAC(SEQ ID NO:3)(284) and IHIGPGRAC (SEQ ID NO:4)(285). Table 27, below,shows the percentages of inhibition of HIV for each vaccine, ascalculated against the control serum.

TABLE 27 Inhibition of HIV for various peptides coupled to KLH SerumDilution 1:25 1:50 1:100 1:200 HIV-MN Control 193,500 182,400 193,600243,200 Control Serum 139,700 152,400 140,900 179,300 282 25,370 (81.9%)25,770 (83.1%) 19,170 (86.4%) 214,300 (19.2%) (GPGRAF)3C 283 20,970(85.1%) 27,470 (82.0%) 28,660 (79.7%) 128,800 (28.2%) IYIGPGRAC 28429,820 (78.7%) 91,620 (72.7%) 95,100 (32.4%) 191,900 (7.0%) IAIGPGRAC285 41,340 (70.5%) 108,000 (29.2%) 177,100 (26.0%) 215,900 (20.4%)IHIGPGRAC

FIG. 9 represents the results of immunization of rabbits with variouspeptides coupled to KLH. The symbols (W, O, F and °) represent thepeptides with which the rabbits were immunized. The abscissa representsthe peptide with which the ELISA plate was coated and the concentrationof peptide in the well. Reactivity with low peptide concentrationrepresents high affinity antibody. FIG. 10 represents the results ofimmunization of rabbits with various peptides coupled to KLH. Theabscissa represents reciprocal rabbit serum dilution reactivity with apeptide-(amino acid sequence shown on the top of the graph) coated ELISAplate with optimal peptide concentration.

EXAMPLE XIII

Immunization of Guinea Pigs with Conjugates of MN-PND Coupled to PPD orToxin A

HIV-1 MN strain obtained from the AIDS Reference and Reagents-Programswas propagated in H9 cells. H9 cells were grown on RPMI (BRL-Gibco,Gaithersburg, Md.) and heat-inactivated fetal calf serum. Cells weregrown in 75 cm² flasks (Corning Glass Works, Corning, N.Y.) in 5% CO₂ at37° C. in a humidified incubator. Cell-free virus particles wereobtained by centrifuging H9 cells chronically infected with HIV-1_(MN)for 30 minutes at 4° C. The cell-free supernatant was collected,filtered, aliquoted, and stored at −70° C. Virus infectious titer wasdetermined by incubating quadruplicate samples of serially diluted viralsupernatant in 1.5 ml of media containing 2×10⁵ H9 cells/well. Afterculturing for 18 days, the P24 content of the supernatant was determinedusing a commercial antigen capture assay (DuPont). The highest dilutionof the virus stock that yielded a productive infection in 50% of thewells was defined as the tissue culture infective dose 50 (TCID₅₀). Forneutralization assays, guinea pig sera was heat-inactivated at 56° C.for 30 minutes. The sera was diluted (1:25 to 1:1500 final dilution) ingrowth medium and incubated with 100 TCID₅₀ of HIV-1_(MN) for 2 hours at37° C. and then added to cultured H9 cells (2×10⁵/well). Controlsincluded growth medium without sera and similarly diluted normal guineapig serum. Following 18 days of cultivation, an aliquot of cell-freeculture supernatant was assayed for P24 antigen content. Control wells(no added serum) contained between 180 to 213 ng of P24/ml, while wellswith normal guinea pig serum contained 180 to 253 ng of P24/ml.Neutralizing capacity was determined by comparing the P24 antigencontent of the test serum samples with those which contained anidentical dilution of control serum. Titer is defined as the reciprocalof the highest dilution of serum which effected a ≧90% decline in P24content. Table 28, below, shows the antibody response after immunizationwith MN-PND-PPD or MN-PND-toxin A conjugate vaccines.

TABLE 28 Anti-MN-PND IgG antibody response after immunization withMN-PND-PPD or MN-PND-TA conjugate vaccines Geometric mean ELISA titer(range) Vaccine BCG Priming Day 0 28 56 84 PND-PPD No <2 <2 2.4 (<2-19)75 (10-5064) Yes <2 8.3 (4-35) 1845 (357-5456) 9035 (7968-19,712) PND-TAYes <2 53 (8-393) 624 (270-1744) 149 (39-309) PND-PPD Yes <2 17 (<2153)957 (250-2040) 599 (158-1300) + PND-TA

Guinea pigs (groups of 5-6) were immunized on days 0, 14, 28, and 56with 1 mg of the PND-PPD conjugate (equal to 0.65 mg of MN-PND peptide)or with 50 mg of the PND-TA conjugate (equal to 10 mg of MN-PND peptide)by the intramuscular route. Where indicated, animals were primed byvaccination with 10⁷ BCG 2 weeks prior to receiving the first dose ofvaccine.

IgG antibodies to the MN-PND peptide were measured by ELISA as follows.To each well of 96-well microtiter plate. was added 100 ml of a 5 mg/mlsolution of peptide in 0.1 M NaHCO₃ (pH 9.6). The plates were incubatedovernight at room temperature. After coating, the liquid was aspiratedand unbound sites were blocked by the addition of phosphate bufferedsaline containing 0.1% (wt/vol) casein, 0.05% Tween 20 and 0.0004%(wt/vol) rhodamine. The test sera were serially diluted in the abovebuffer and 100 ml added per well. After incubation for 1 hour at 37° C.,the plates were washed and peroxidase-labeled anti-guinea pig IgG wasadded. After incubation for 1 hour at 37° C., the plates were againwashed and substrate added. The absorbance at 405 nm was measured after30 minutes of incubation at room temperature using a Titertek Multiscan(Flow Laboratories, McLean, Va.). Serum samples from the same animalwere run in parallel on the same plate. On each plate were also runpositive and negative control sera. Titers were determined bymultiplying the optical density (OD) of a serum sample which fell withinthe linear range (A₄₀₅ 0.2-0.8) by its reciprocal. Table 29, below,shows recognition of heterogenous PND peptides and GPGRAF motiffollowing immunization with the MN-PND-PPD and MN-PND-Toxin A conjugatevaccines.

TABLE 29 Recognition of heterologous PND peptides and GPGRAF motiffollowing immunization with MN-PND vaccines Peak geometric mean PNDpeptide Peak geometric antibody affinity PND-PPD (strain of HIV-1) meantiter (ng peptide/ml) PND-PPD KRIHIGPGRAFYT (MN) 9035  20 KSIHIGPGRAFYA(SC) 1045  301 KSITKGPGRVIYA (RF)  11 3465 KGIAIGPGRTLYA (NY-5)   6 2201SRVTLGPGRVWYT (CDC42)   3 7578 GPGRAF³  478  397 PND-TA KRIHIGPGRAFYT(MN)  624  71 KSIHIGPGRAFYA (SC)  171  89 KSITKGPGRVIYA (RF)  10 8908KGIAIGPGRTLYA (MY-5)  34  500 SRVTLGPGRVWYT (CDC42)   3 10,000   GPGRAF³1581  500 PND-PPD KRIHIGPGRAFYT (MN)  957  56 + KSIHIGPGRAFYA (SC)  500 89  10 PND-TA KSITKGPGRVIYA (RF)  58 10,000   KGIAIGPGRTLYA (MY-5)  451523 SRVTLGPGRVWYT (CDC42)  12 5407 GPGRAF³ 3623  416

Non-antigen limited and antigen limited ELISA to detect total anti-PNDantibodies and high affinity anti-PND antibodies, respectively, wereperformed. Table 30, below, shows in vitro neutralization of the MNstrain of HIV by guinea pig sera.

TABLE 30 In vitro neutralization of the MN strain of HIV by guinea pigsera Geometric mean neutralizing Vaccine titer (Range) MN-PND-PPD <25MN-PND-TA 733 (500-1500) MN-PND-PPD 487 (500-1500) + MN-PND-TA

FIG. 11 shows maturation of high affinity anti-MN-PND antibody response.An antigen limited ELISA was used to detect high affinity antibody.ELISAs were performed as described, with the exception that each ofeight microtiter plate rows were coated overnight by the addition of5,000, 1,000, 500, 100, 50, 10, 5, or 0 ng peptide/ml (100 ml/well).Serum samples were diluted 1:20 and 100 ml added per well. Antibodyaffinity was assigned the value of the lowest concentration of peptidewhich gave an absorbance ≧5 standard deviations above the negativecontrol serum. Animals (groups of 5-6) were vaccinated on days 0, 28,and 56 with PND-PPD (o), PND-TA (″), or PND-PPD-plus PND-TA (W).Immunization with the MN-PND-Toxin A vaccine induced the production ofantibodies capable of neutralizing HIV MN in vitro.

EXAMPLE XIV

Immunization of Guinea Pigs with Conjugates of Peptides Coupled to PPD

A polyvalent vaccine was formed by conjugating peptides from theprincipal neutralizing domain of various strains of HIV to the carrierPPD. Specifically, peptide KSIYIGPGRAFHT (SEQ ID NO:7) from HIV strainARV-2, peptide SRVTLGPGRVWVYT (SEQ ID NO:9) from HIV strain CDC-42,peptide KRIHIGPGRAFYT (SEQ ID NO:1) from HIV strain MN, peptideKGIAIGPGRTLYA (SEQ ID NO:8) from HIV strain NY-5 and peptideKSITKGPGRVIYA (SEQ ID NO:7) from HIV strain RF were each coupled to thecarrier PPD. After conjugating each of the 5 peptides to PPD, 3 mg ofeach conjugate was combined to obtain a 15 mg polyvalent vaccine.

First, 6 guinea pigs were A primed with BCG. After priming the guineapigs, each guinea pig was injected with 5 mg of the polyvalent(pentavalent) vaccine. At a later date, sera from the vaccinated guineapigs was analyzed in an ELISA of the invention.

Table 31, below, shows the GM ELISA titer for all 5 vaccine components.Sera was assayed at days 0, 42, 56 and 78. All 5 of the vaccinecomponents elicited a significant antibody response when compared tobaseline values. As shown in Table 31, the KRIHIGPGRAFYT peptide fromthe MN strain of HIV was the most immunogenic and KSITKGPGRVIYA peptidefrom the RF strain of HIV was the least immunogenic. This data showsthat multiple monovalent conjugates of this invention can be combined toyield an immunogenic multivalent vaccine.

TABLE 31 Immunogenicity of Polyvalent PND-PPD Conjugate Vaccine inGuinea Pigs GH ELISA TITER VACCINE DOSE DAY ARV-2 CDC-42 MN MY-5 RFTHAI-2 PND-PPD   3 ug  0 <2 <2 <2 <2 <2 <2 (289) 42 15  6 14 26  8  5 56551  148  745  852  164  31 78 5773  2780  7342  8571  5163  1441 PND-PPD 0.5 ug  0 <2 <2 <2 <2 <2 <2 (289) 42  4  4  5  1  4  2 56 26 2327 51 16  9 78 1068  740  2302  3869  568  122  Groups of guinea pigs[5-6] were primed with 10⁷ BCG 2 months before immunization. PND-PPDimmunizations on days: 0, 14, 28, 42 and 56.

EXAMPLE XV

Peptides Used in the Multiepitope PPD/10 kMtb Vaccines

V3 loop peptides. Linear peptides from amino acids 307-319 associatedwith the V₃ loop region were synthesized as set forth in Devash, et al.Proc. Natl. Acad. Sci USA 1991; 87:345-49 and Kollman, et al. Proc.Natl. Acad. Sci. 1996; 93:3126-31. No longer than 18 amino acidsequences were prepared which represented THAI-I, THAI-II, MN RF, NY-5,CDC-42, ARV-2 and a representative Brazilian strain were synthesized.

gp41 peptides. The 6 amino acid peptide ELDKWA shown by Katinger(Muster, et al. J. Virol. 1994; 68:4031-34) to be the target of across-neutralizing monoclonal antibody with two additional LL (LLEDKWA)(SEQ ID NO:10) was synthesized as a single motif and a repetitive motifof 16 amino acids. The peptide sequence without the additional two LL isnon immunogenic.

Nef peptides. The selection of nef epitopes is based on the conservedfeatures of nef sequences, on their functional properties (Nixon, D. F.,et al. AIDS 5:1049, 1991; Cheingsong-Popov, R., et al. AIDS Res. & HumanRetrov. 6:1099, 1990; Schneider, et al. AIDS Res. & Human Retrov. 1:37,1991; Siakkou, H., et al. Arch. Virol 128:81, 1993; Culmann, B., et al.J. Immunol. 146:1560, 1991; Yu, G., et al. Virology 187:46, 1991;Shugars, D. C., et al. J. Virol. 67:4639-4650, 1993; Venet, A., et al.AIDS Res. & Human Retrov. S41, 1993; Robertson, M. N., et al. AIDS Res.& Human Retrov. 9:1217: 23, 1993) and on sequences found missing inpatients with “non-virulent” HIV-1 disease (long-term non-progressors).Sequences known to induce both humoral and CTL responses(Cheingsong-Popov, R., et al. AIDS Res. & Human Retrov. 6:1099,. 1990;Schneider, et al. AIDS Res. & Humra Retrov. 1:37, 1991; Siakkou, H., etal. Arch. Virol 128:81, 1993; Culmann, B., et al. J. Immunol. 146:1560,1991; Robertson, M. N., et al. AIDS Res. & Human Retrov. 9:1217: 23,1993) are selected. Other peptides that may be employed include, forexample:

a. RPMTYK (SEQ ID NO:11)—a highly conserved recognition site forphosphorylation by protein kinase C:

b. GGKWSK (SEQ ID NO:12)—a nearly invariant myristilation site whichlies on the external surface of the folded nef protein.

c. PGPGIRY (SEQ ID NO:13) and GPGIGPGV (SEQ ID NO:14) located atpositions 13-138, a highly conserved region predictive of a beta turn(Shugars, D. C., et al. J. Virol. 67:4639-4650, 1993).

Carriers for the PND peptides. PPD: several lots from differentmanufacturers and the same manufacturer were tested for immunogenicity.Recombinant proteins from M. tuberculosis and M. leprae: Cloning andexpression of the genes of the secreted 10 kDa and 32 kDa from M.tuberculosis and M. leprae were synthesized. Linear co-expression of 10kDa and PND yielded a 1:1 ratio between both components resulted in poorimmunogenicity. Conjugation of peptides to 10 kDa M. tuberculosis and to10 kDa M. leprae using glutaraldehyde allowed a 4-5:1 peptides/proteinmolar ratio could be conjugated to each protein molecule. Toxin A fromPseudomonas aeruginosa: Toxin A (TA) was irreversibly detoxified bycovalently coupling to adipic acid dihydrazide and allowing haptens tobe coupled through the nonreacted hydrazide groups (Lussow, et al. Proc.Natl. Acad. Sci. USA 1990; 87:2960). Filamentous Hemagglutin (FHA) ofBordetella permusis: PND peptides were covalently coupled to FHA usingthe ADH/Carbodimide reaction at a molar ratio of 10-12:1. T helper cell(Th) epitopes of tetanus toxoid (TT): Conjugation of HIV peptides to Thelper cell epitopes of tetanus toxoid was performed. Several peptidesfrom the TT described to be universal Th cell epitopes recognized by themajority of the human population. were synthesized. TT Th epitopes andHIV peptides were chemically coupled to liposomes or virosomes.Glucoconjugate-with synthetic peptides: Capsular polysaccharide fromGroup B streptococcus and Dextra T-500 were coupled by reductiveamination in the presence of sodium cyanoborohydride to PND-NY5-PND, andto 10 kDa M. tuberculosis NY5-PND at different ratios.

EXAMPLE XVI

Animal Immunization

Immunogenicity of PPD-Mn-PND conjugates. Guinea pigs were primed byintramuscular 10⁷ CFU of BCG 14 days prior to the first PND vaccination.BCG priming was required to obtain an anti-Mn-PND antibody response. Thelowest dose of peptide capable of engendering. an immune response wastitrated. BCG-primed guinea pigs were immunized with 0.5-3.0 μ6 PPD PND.Following five doses of 1 μg PND (day 98) there was a greater than100-fold rise in anti-Mn-PND antibody titer. In contrast to the TA-PND,the peak GMT antibody titer of 9,035 was achieved with the PPD-PNDconjugate only after 3 immunizations. These antibodies possessed a veryhigh mean affinity for the MN-PND peptide, cross- reacted with PNDpeptides from SC, RF, NY5, neutralized HIV-1 MN prototypes at highertiters than (1:3600) TA-PND but also failed to neutralize primaryisolates.

Hexa-PND-PPD conjugates. Guinea pigs were immunized with either 0.65microgram of MN-PND-PPD or with 3.0 microgram of the hexa-PND-PPDconjugate mixture (0.5 microgram per peptide). Table 1 shows thegeometric mean titer (GMT) of antibodies to the primary neutralizingdomain (PND) in guinea pigs immunized with a hexa-PND-peptide-PPD versusa mono pnd (MN-peptide-PPD vaccine. The GMT of antibodies to thehexa-peptide vaccine were about 15 fold higher and more broadlycross-reactive as compared to- the mono-peptide vaccine. Furthermore,the hexa-peptide induced for the first time neutralizing antibodies ofhigh titer also to primary isolates (Table 32). The PPD hexa-PND vaccinealso induced neutralizing antibodies for primary HIV-1 isolates in PBMCat 1:45-1:135 titers (see Table 33).

TABLE 32 Immunogenicity (GMT) of 3 μg of a hexa-peptide PND-PPDconjugate vaccine (0.5 g per peptide) versus 0.65 μg of a mono-peptide(MN) PND-PPD conjugate vaccine. Mono-PND peptide Hexa-PND-peptide MN-PPDConjugate PPD Conjugate Day Post-Vaccine MN MN RF NY5 ARV-2 CDC-2 THAI-2 0 <2  <2  <2  <2  <2  <2 <2 42 2.4  14  8  26  15  6  5 56 121.1 745164 852 551 148 31 78 462 7342  5163  8571  5163  2780  1441  Groups of5-6 Guinea pigs were primed with 10⁷ BCG 2 months before immunization.Peptide immunizations were on days: 0, 14, 28, 42 and 56. Note: themono-peptide PND conjugate vaccine induced cross-reactive antibodieswith a GMT of s20 to RF, NYS, ARV-2, CDC-42 AND THAI-2 (not shown) ascompared to GMT of up to 8,571 in the HEXA-PND-PPD vaccine.

TABLE 33 Neutralization of 100 TCID₅₀ of HIV-I-28 (primary patientisolated) in PBMC assay by sera from guinea pigs (gp) immunized with thehexa-PND-PPD vaccine (pg p24/ml). Immunized gp Serum Dilution gp # 1:151.45 1:135 1:405 1:1,215 1:3,645 Control 9323 0.1* 0.1 2,728 2,624 6,8246,824 4,144 9325 0.1 0.1 0.1 2,595 2,873 3,545 4,114 9326 0.1 0.1 0.11,025 2,725 3,061 4,114 9333 0.1 124 538   558   639 1,443 4,114 93360.1 2,282 2,134 2,314 3,026 3,254 4,114 9337 0.1 0.1 1,106 1,830 3,5242,456 4,114 BCG primed gp were immunized on days 0, 14, 28, 56. Seratested were drawn on day 78. *pg p24/ml. Note that in 4 of 6 gp there issignificant neutralization of the primary isolate up to a dilution of1:135. In gp 9333 there is neutralization up to a 1:3645 serum dilution.With the addition of fresh gp complement in one tested guinea pig theneutralization titer was increased about 100 fold in one tested guineapig.

Immunization with 10 kDa and 32 kDa M. tuberculosis and M. lepraeconjugates of MN-PND: These conjugates yielded the highest antibodytiters after 3 to 8 monthly injections in BCG primed animals. Antibodytiters were, however, about 5-10 fold lower than with PPD-MN conjugates.

Immunization of mice with NY5-PND coupled to 10 kDa M. tuberculosis orto PPD and to capsular Polysaccharides from group B Streptococcus 1b andDextran T-500. BCG primed mice were immunized at days 0, 14, 28 and 91.Moderate antibody titers were obtained.

Immunization of mice with FHA and with tetanus T-helper epitopes (ThTT)with and without virosome or liposome formulations have shown resultsinferior to PPD-MN-PND (not shown).

Immunizations of mice with 10 kDa M. tuberculosis conjugates with nef,gp41 have shown unimpressive antibody titers to gp41 and moderate titersto nef peptides. These antibodies were, however, of high affinity andneutralized primary isolates at 1:25 dilutions.

Synergistic, sequential immunizations. Three monthly immunizations ofguinea pigs with 10 kDa M. tuberculosis MN-PND and nef 10 kDa M.tuberculosis peptide mixture conjugate (animal 2521) or with MN-PND 10kDa+gp41 10 kDa M. tuberculosis conjugate also resulted in moderateantibody titers to the respective peptides and both induced impressiveconsistent neutralization of primary HIV-1 isolates in PBMC (see Table34).

TABLE 34 Neutralization of HIV-1 prototype an a primary isolates byguinea pigs immunized with MN-10 kDa M. tuberculosis followed bypenta-Nef peptide-10 kDa M. tuberculosis, respectively, gp-41 peptide 10kDa M. tuberculosis (pg p24/ml) gp 2521 gp 2517 20 μg 10 μggp41-peptide- gp 2501 Nef-peptide-10 kDa 10 kDA MTB 5 μg gp41 Serum HIVprimary HIV primary HIV primary dilution prototype isolate prototypeisolate prototype isolate Control 58,643 35,516 38,790 33,303 73,746  646 1:15  4,702  2,303  3,637  2,528  3,929    8 1:45  9,970 12,477 7,097  8,681  7,070   12 1:135 43,787 20,047 34,572 21,002 29,115   101:405 47,274 20,283 69,722 30,825 71,828   25 1:1,215 85,890 40,14774,631 40,943 68,790 1,206

HIV-1-MN prototype of 50 TCID₅₀ was tested in 119 target cells. HIV-1-59primary isolate 200 TCID₅₀ was tested in PBMC immunization schedule: allgp were immunized intradermally with 50 μg of MN PND-10 kDa on days 0,32, 62, 90 and 114. No primary isolate neutralizing antibodies wereinduced by this vaccine. Animals were rested to day 227. 2 animals werethen immunized on day 227 and 270 with 5 μg, respectively, 10 μg ofpeptide 10 kDa, and one animal was immunized with four-Nef-peptide-10kDa Mtb conjugate. Bloods were drawn on day 305. Note that in all 3animals there was modest neutralization of both HIV prototypes andprimary isolate. The studies in gp 2501 with primary isolate have to berepeated.

Immunization of HIV infected PPD skin test positive volunteers with thehexa-PND-PPD conjugate vaccine. 7 patients with CD4 cells above 200 wereselected. All patients had a positive skin test to PPD of 5 mm diameteror greater. Patients were immunized with 3.0 microgram of the 6 peptidemixture on days 0, 30, 90, 120, 180 and 240. All patients remainedclinically stable over a period of 1 year, the CD4 cells increased in 3and remained stable in the others. All patients developed extremely hightiters (265 fold) of neutralizing antibody titers to primary isolates(FIGS. 16A and 16B and FIG. 17), a representative log reduction assayfor HIV-1 primary isolates in one patient. The same pattern was shown inall patients receiving this vaccine by 12 months). No other vaccine hasso far achieved such a remarkable immune response.

EXAMPLE XVII

Reduction in HIV Viral Load and Improved Neutralization of HIV-1 PrimaryIsolates

Patients: Asymptomatic HIV-1 seropositive subjects were enrolled fromthe HIV clinic of the University of Tel-Aviv, Elias Sourasky MedicalCenter. Patients had to have a positive intradermal skin test to 2tuberculin unit (TU) PPD (induration and/or erythema of ≧5 mm after 48hours). Laboratory eligibility criteria included routine laboratoryvariables (CBC, chemistries) within the normal ranges, a CD4+ cellcount >250 cells/ml. Exclusion criteria included the use ofantiretroviral combination therapy and any disorder meeting the USA 1987Centers for Disease Control AIDS surveillance definition.

Seven patients, ages 24 to 46 who were PPD skin-test positive at the 2TU intradermal dose were classified as eligible for the protocol. Twopatients (#4 and #5) were on reverse transcriptase inhibitor monotherapyprior to and during vaccination (Table 35).

TABLE 35 Clinical status, CD4 cell count and viral load of HIV+, PPD+vaccinees prior to immunization with the PPD-pentapeptide-PND conjugate.Pa- Clinical Antiretrovirals Viral load tient Symp- during CD4 PCR-RNA #Sex Age toms immunizations cells/cmm copies/cmm 1 F 40 Asymp- None 5005,700 tomatic 2 M 32 Asymp- None 620 15,000  tomatic 3 M 46 Asymp- None260  <400 tomatic 4 M 28 Asymp- ddI 491  <400 tomatic 5 M 38 Asymp- ddc440 12,000  tomatic Pso- riasi- form 6 F 26 Asymp- None 490  <400tomatic 7 F 24 Asymp- None 450 1,100 tomatic

The vaccine protocol was approved by an independent ethical committee atthe University of Tel-Aviv, Israel and received approval according tothe Helsinki Committee. Approval to utilize study subjects' specimen fortesting at the Albert. Einstein College of Medicine was obtained fromthe University's Investigational Review Board.

Vaccine synthesis and characterization: PPD obtained from the StatensSerum Institute, Copenhagen, Denmark was a solution containing 1 mgPPD/ml equal to 50,000 TU/ml (1 μg PPD=50 TU).

V₃ loop peptides of the following sequences (representative for the HIVvariant in parentheses), KRIHIGPGRAFYT (SEQ ID NO:1)(MN), RSIHIGPGRAFYA(SEQ ID NO:6)(ARV-2), KGIAIGPGRTLYA (SEQ ID NO:8)(NY-5), KSITKGPGRVIYA(SEQ ID NO:7)(RF), SRVTLGPGRVWYT (SEQ ID NO:9)(CDC-42), were prepared bystandard solid phase synthesis. Peptide sequences were verified byanalysis on an automated amino-acid analyzer and by high pressure liquidchromatography (HPLC).

The PPD-peptide conjugation was prepared with gluteraldehydes aspreviously reported. (Rubinstein A, et al. AIDS 1995; 9: 243-51). Fivemonovalent PPD-PND conjugates were combined in equal amount (based uponPPD contents) to yield the final vaccine which contained 50 TU PPD and0.13 μg of each peptide or a total of 0.65 μg of the five V₃ looppeptides listed above.

Immunization Schedule: Participants were scheduled to be immunizedmonthly for the first 3 months and then at 3 month intervals to 18months. Only patients #1-3 completed the whole regimen. Patients #4 and#6 received the last immunization at 12 months, patient #7 at 9 monthsand patient #5 at 6 months. All patients were followed for 18 monthsfrom entry to the study. The vaccine was given intradermally 25 TU PPDinto each arm. All vaccinees were monitored for local and systemicreactions. A standard battery of complete blood counts and bloodchemistry profiles were performed periodically. Blood specimen forresearch studies were all coded. The decoding of clinical and laboratorydata was conducted only at the conclusion of the study.

V₃ loop enzyme-linked immunosorbent assay (ELISA): Microtiter ELISAplates (Nunc, Naperville, Ill., USA) were coated with the respectivesynthetic V₃ loop peptide and incubated with patient sera diluted 1:20as previously reported. (Rubinstein A, et al. AIDS 1995; 9: 243-51;Devash Y, et al. Proc Natl Acad Sci USA 1991; 87: 345-49).

Antigen-limited ELISA: Microtiter ELISA plates were coated with avaccine peptide or with (GPGR)₃AF at decreasing concentrations from10,000 ng/ml to 10 ng/ml, as previously reported. (Rubinstein A, et al.AIDS 1995; 9: 243-51; Devash Y, et al. Proc Natl Acad Sci USA 1991; 87:34549). An optical density (OD) above background at ≦100 ng/ml wasconsidered to indicate affinity.

Virus Neutralization assays: (A) Infectivity reduction assay: Theinfectivity reduction assay estimates the reduction in the number ofinfectious units per/ml (IUPML) when treated with patient sera andprogressive dilutions of viral stocks, according to the NIAID AIDSClinical Trial Group consensus protocol. The assay was performed in24-well plates using 5-fold dilutions of viral isolate (1:5 to1:390,625). Each 1:20 diluted serum sample was tested with a stocked,previously tittered inoculum of clinical isolates as reference virus(e.g. MN referenced, primary isolate HIV-1-59) or with autologous virus.The virus stock's TCID₅₀ was determined by the method ofSpearman-Karber. Uniformity of donor target cells was secured byfreezing sufficient PBMC's from a panel of previously tested donors.PBMC's were activated for 48 hours with PHA/IL-2 prior to use. Eachsample of viral dilution was cocultured at 37° C. with activated donorPBMC (1×10⁶) for 14 days. Concurrent, neutralization controls and virustitration controls were performed in the same plate. On day 7 half ofthe medium was removed and saved for future testing, and replaced withfresh medium containing 0.5×10⁶, 48 hours PHA/IL2 stimulated normaldonor PBMC's. Cultures were terminated on day 14, and collectedsupernatant was tested for total p24 content. Infectivity reduction isexpressed as the ratio of log1D₅₀ for the control and test serum (foldinhibition). A 0.3 log reduction was considered significant. (B) Restingcell assay was performed according to Zolla-Pazner and Sharpe.(Zolla-Pazner S, et al. AIDS Res Hum Retrov 1995; 11: 1449-58). A CladeB non-syncytium-inducing virus BZ167 was kindly provided by Dr.Zolla-Pazner. In addition, the Clade B primary isolate HIV-1-59 wasused. Virus stock was diluted to 100 TCID₅₀. Serial serum dilutions werethen added. to the wells. The percent inhibition by serum was calculatedfrom the p24 counts at 1:625 serum dilutions of baseline serum ascompared to a 12 month post immunization serum. An inhibition of morethan ≧50% was considered significant.

Viral load determination: This study was performed in duplicates with acommercial assay (NASBA; Organon, Teknika; Durham, N.C.), in which thelower limit of detection is 400 RNA-copies. Frozen specimen were batchedand tested simultaneously. The intra-assay variability was 0.12-0.2logs. Sustained changes in plasma HIV RNA levels greater than three foldover time were considered relevant. (Saag MS, et al. Nature Medicine1996; 2: 625-29).

CD4 cell counts: CD4 cells were measured by flow cytometry by standardmethods as previously described. (Devash Y, et al. Proc Natl Acad SciUSA 1991; 87: 345-49).

Results

The monovalent MN-peptide-PPD vaccine failed to induce in HIV-1 negativevolunteers neutralizing antibodies to primary virus isolates, incontrast to the polyvalent vaccine (See Table 35) which induced longlasting. immunity with neutralizing antibodies to HIV-1 primaryisolates, utilizing the novel principle of synergistic immunization.Immunization. with 10k and 30k M tuberculosis (Mtb) and M lepraeconjugates with Hexa-PND conjugates showed similar results as for thePPD conjugates. Immunizations with hexa PND+gp41+nef peptide conjugateshas yielded broader immune responses in guinea pigs than with any of theprevious conjugates. Sequential immunizations have also yielded superbresponses with consistent impressive neutralization of primary HIV-1isolates.

Vaccine acceptability: A total of 64 vaccine doses were administered. Nosystemic reactions or laboratory abnormalities were noted. Typical localreactions at the intradermal vaccine site were redness and indurationwithin 24-48 hours, which resolved within 2-4 days.

Antibody responses to the vaccine: By 3 months an increase in antibodytiters to all 5 vaccine peptides and to the GPGRAF motif was noted inall patients (FIGS. 12A-12G). The antibody titer increased withadditional immunizations and was maintained in all patients except forpatients #5 and #7 who received the shortest course of vaccination (6months, 9 months, respectively).

Affinity of serum antibodies: High affinity antibodies to vaccinepeptides were noted in all vaccinees after the third boost (not shown).There was a further increase in affinity over time in all patientsexcept in patients #5 and 7 who had the shortest vaccination course: inpatient #5 (FIG. 13B) and #7 the affinity increased remarkably at 3months (to an OD of 0.807 at the 10 ng/peptide well) but dropped back tobaseline at month 12.

CD4 cell count: Overall, there was no dramatic change in CD4+ cellcounts over the 18 months observation period. Five patients showed adecline ranging from 10 to 170 cells while 2 showed an increase (40,respectively 160 cells). Maximal increases or decreases over the studyranged from +340 to −170 (Table 5). There was no correlation between anincrease or decrease in CD4+ counts and viral load.

TABLE 36 CD4+ cell counts/cmm post immunization withPPD-PND-pentapeptide conjugate. Maximal Net Change at increase 15-18months (decrease) over baseline Patient Months Post Immunization CD4 CD4# 0 3 6 9 12 15 18 cells/cmm cells/cmm 1 500 420 450 450 410 450 486 −90 −14 2 620 600 640 — 580 560 450 −170 −170  3 260 390 380 420 420240 220 +160 −40 4 490 350 470 432 660 825 650 +335 +160  5 440 500 480— 496 475 480  +60 +40 6 490 640 530 520 527 550 480 +150 −10 7 450 530790 — 520 424 — +340 −26

Virus neutralization: After 3 months of immunization there was a2.7-10.8 fold increase in the HIV neutralizing antibody over baseline(Table 37; FIGS. 14A-14G). With subsequent doses of vaccine there was asubstantial increase (36-172 fold) in neutralizing activity with theexception of patient #7 (Table 35; FIGS. 14A-14G). In patient #7 therewas a decrease in the neutralization titer after discontinuation ofimmunizations. In. three out of four tested patients there was also asignificant increase in neutralization of autologous virus (Table 37).Patient #7 who failed to neutralize autologous virus also did notneutralize primary isolates. Two patients (#2, #5) tested in the restingcell assay showed significant neutralization of primary isolates (Table37).

TABLE 37 HIV-1 infectivity reduction (neutralization) assay* and restingcell neutralization assay** post vaccination with thePPD-PND-pentapeptide conjugate. % HIV-Inhibition Fold Inhibitionpost/pre-immunization Resting Cell HIV-1 Infectivity reduction assayAssay Primary Isolate Autologous Virus Primary Isolate Patient # 6months 12 months 12 months 12 months 1 2.7 108 nd nd 2 5.0 53 20   80% 34.8 53 17   nd 4 3.2 172 nd nd 5 7.1 36 nd 84% 6 10.8 53 18   nd 7 3.01.4  1.0 nd *Using the NIAID-ACTG consensus log reduction assay. TheTCID50 reduction was calculated according to the Spearman Kraber method.**According to Zolla-Pazner AIDS Res & Human Retrov 1995; 11:1449. nd =not done

Virus Load: The viral load remained <400 copies throughout the studyperiod in those patients who were negative at entry (#3 & #6; FIGS.14A-14G). In the three patients (#1, #2 & #5) with substantial viralloads at enrollment, there was a progressive virus decline noted whichlasted for at least 15 months. In patients #2 and #5, the viral load haddecreased to <400 copies during the course of the study. In patient #4and #7, who had low viral burdens at entry, vaccination effected areduction to non-detectable limits. There was a clear trend towards anincrease in viral loads in patients who did not receive routine boosterdoses of vaccine. For example, in patient #5 (last does of vaccinationat 6 months) immunization effected a reduction in viral load from 14,000copies to <400 copies by 9 months. While the viral load remained atundetectable levels from month 9 to 15, it increased dramaticallyto >18,000 by 18 months. A similar picture was seen in patient #7 whowas last vaccinated at 9 months. By 9 months the viral load had droppedfrom roughly 1,200 to undetectable where it remained through month 12.However, by month 15 the viral burden had increased to 13,000.

Discussion

Relentless and high level of HIV-1 replication are responsible for theimmune attrition in AIDS patients. (Saag M S, et al. Nat Med 1996;625-29; Mellors J W, et al. Ann Intern Med 1995; 122: 573-79; Ho Dd, etal. Nature 1995; 373: 123-126; Fauci A S, Nature 1996; 384: 529-33).Despite the high level of viral replication the immune system issometimes capable of containing HIV-1 infection as is the case in longterm non-progressors. (Haynes B F, Lancet 1996; 348:933-37; Haynes B F,Lancet 1996; 348: 1531-2; Haynes B F, et al. Science 1996; 271: 324-8).Salk et al (Salk J, et al. Science 1993; 260: 1270-72) have suggestedthat immune enhancement in HIV-1 infected individuals may further limitdisease progression. However, studies to support the effectiveness ofvaccines in containing disease progression are inconclusive. Redfield etal (Redfield R R, et al. N Engi J Med 1991; 324: 1667-84) have shown ina subgroup of HIV-1 infected individuals receiving a recombinant gp160vaccine that those who had both a humoral and cellular immune responseto the vaccination had also a slower decline in their CD4 cell counts.It was unclear whether this was actually a result of vaccination.Valentine et al (Valentine F T, et al. J Infect Dis 1996; 173: 1336-46)and Eron et al (Haynes B F, et al. Science 1996; 271: 324-8) have notedthat a recombinant gp160, respectively gp12 vaccine induced in HIV+individuals humoral and cellular immune responses that infection itselfdid not stimulate. Furthermore, immunized subjects made antibodies to anenvelope protein and to p24 both of which were not present in thevaccine suggesting that the vaccine acted both as specific and as a nonspecific immunostimulant. In both trials vaccinations did not decreaseviral plasma load, a main parameter for disease containment.

The main stumbling block in the use of recombinant envelope proteinvaccines was considered to be the continuous emergence of variantviruses within an individual over time and the large variability of theV₃ loop within one clade of HIV-1 and between different clades. (KuikenC L, et al. Proc Natl Acad Sci USA 1993; 90: 9061-65; Matthews T J, etal. Proc Natl Acad Sci USA 1986; 83: 9709-13; Gorny M K, et al. ProcNatl Acad Sci USA 1991; 88: 3238-42). There are, however, indicationsthat this variability is not an insurmountable obstacle. The V₃ regionretains many of its individual characteristics in an infected individual5 years after infection.. (Kuiken C L, et al. AIDS 1996; 10: 31-7).Furthermore, immunization of chimps with recombinant gp160 elicitedanti-V₃ antibodies with broad crossreactivity to various field isolatesdespite their variable V₃ sequences. (Boudet F, et al. AIDS Res HumanRetrov 1996; 12: 1671-79). The peptide vaccines described herein werealso broadly and highly immunogenic probably due to the aggregate ofseveral unique features.

It has been shown that fractional doses of intradermally injected poliovaccine induced an up to 1024-fold increase in antibody titer. (Samuel BU, et al. Lancet 1991; 338; 343-44). The advantage of this route ofvaccine administration may be due to the engagement of ample localdendritic cells as antigen presenting cells capable of priming T cells.This may explain why our intradermally applied vaccine elicited highaffinity antibodies of both the IgG and IgA class and MHC class-II Tcell responses in HIV-1 uninfected vaccinees. (Rubinstein A, et al. AIDS1995; 9: 243-51). It does not, however, fully explain the broadimmunogenicity achieved. The latter may be attributed to the use of PPD,a unique carrier in that everyone with a functional immune response whohas been exposed to tuberculosis will respond with a DTHR to minuteamounts of PPD. This universal T-cell recognition may be due to the factthat PPD is derived from cultures of autolyzed bacteria containing amixture of degraded and preprocessed antigens that can be presented bythe majority of major histocompatibility complex (MHC) class-IIhaplotypes. This may explain why a PPD conjugated peptide fromPlasmodium falciparum elicited antibody responses in geneticallynon-responder mouse strains to this epitope. (Lussov A R, et al. ProcNatl Acad Sci USA 1990; 87: 2960-64). BCG presensitized mice alsoproduced antibodies to peptide and carbohydrate epitopes only if thesewere conjugated to PPD. (Lussov A R, et al. Proc Natl Acad Sci USA 1990;87: 2960-64; Lachman P J, Ciba Fund Symp 1986; 119: 25-57). Furthermore,PPD contains a variety of heatshock proteins that may induce unusualimmune responses including Tcell responses. (Mehra V, et al. J Exp Med1992; 175: 275-84; Barnes P F, et al. J Immunol 1992; 148: 1835-40).

It has yet to be determined whether PPD is responsible for the inductionof MHC class-I (HLA-B7 restricted) responses observed by us in HIV-1uninfected vaccinees receiving a PPD-MN-PND conjugate vaccine.(Rubinstein A, et al. AIDS 1995; 9: 243-51). It has been shown thatovalbumin presented to antigen processing cells together with livetubercle bacilli initially enters the cell vacuole to induce MHCclass-II restricted responses but then also translocates to thecytoplasm leading to MHC class-I restricted responses to ovalbumin.(Mazzaccaro R J, et al. Proc Natl Acad Sci USA 1996; 93: 11786-11791).Live mycobacteria may secrete a pore forming hemolysin, similar tolisteriolysin, (Barry R A, et al. Infect Immun 1992; 60: 1625-32) thatallows escape of antigens from the vacuole into the cytoplasm as analternate MHC class-I antigen processing pathway. It is possible that apore forming factor is present in PPD, a crude extract of thesupernatant of mycobacterial cultures.

Using the monopeptide, PPD-MN-PND vaccine in HIV-1 negative subjects,their sera neutralized HIV-1 prototypes but failed to neutralize primaryHIV-1 isolates as also reported for other envelope subunit vaccines.(Mascola Jr, et al. J Infect Dis 1996; 173: 340-48). Haynes et al(Haynes B F, et al. J Immunol 1993; 151: 1646-53) have shown that amixture of HIV-1 envelope peptides can induce in animals antibodiescapable of neutralizing a broad range of HIV-1 isolates. It was observedin guinea pigs immunized with a panel of five V₃ loop peptidesconjugated to PPD (PPD-pentapeptide-PND) an over 10 fold increase inspecific antibody responses to individual PND peptides as compared toimmunization with single PND peptide PPD conjugates. Moreover, only thepentapeptide vaccine induced antibodies that also neutralized primaryisolates in PBMCs (not shown). Based on these results we have embarkedon the present study with a PPD-pentapeptide-PND vaccine in HIV-1infected volunteers with remarkable immunological and virologicalimprovements.

After 3 monthly immunizations, there was already an increase in antibodyresponses noted to vaccine peptides noted (FIGS. 12A-12G). In addition,all sera exhibited an augmented response to the V₃ loop apex (GPGR)₃AF(FIGS. 12A-12G). In animals, the GPGRAF sequence was shown to be thetarget of neutralizing antibodies resulting from peptide immunization.(Boudet F, et al. AIDS Res Human Retrov 1996; 12: 1671-79; White-ScharfM E, et al. Virology 1993; 192: 197-206; Javaherian K, et al. Science1990; 250: 1590-93). The ability of our vaccine to rapidly induce suchantibodies may, therefore, be of clinically relevant.

Similar to the results with the monovalent PPD-MN-PND vaccine in HIV-1negative volunteers, we have also observed with the PPD-pentapeptide-PNDimmunization of HIV-1 infected individuals a marked increase in antibodyaffinity after 3 months (FIGS. 13A and 13B). The affinity increased withadditional immunizations (FIG. 13A, patient #2). However, in patient #5who received his last immunization at 6 months, the antibody affinitybut not the antibody titer returned to baseline by 12 months. Six monthsafter the loss of high affinity antibodies this patient also exhibited arise in viral titers. In HIV-1 infection the spontaneously occurringantibody response requires 8-18 months to reach avidity maturation(Thomas H U, et al. Clin Exp Immunol 1996; 103: 185-91) which ischaracteristic of many T-dependent responses. Using the vaccinedescribed herein, this response occurred much earlier, within 3 months(FIGS. 13A and 13B). The level of in vitro neutralization of primaryisolates by monoclonal antibodies for the V₃ loop was shown to correlatewith the affinity of the antibody (Zolla-Pazner S, et al. AIDS 1992; 6:1235-47) and a correlation has been shown of antibody affinity withHIV-1 maternofetal transmission. (Rubinstein A, et al. AIDS 1995; 9:243-51; Devash Y, et al. Proc Natl Acad Sci USA 1991; 87: 345-49). Theenhancement of antibody affinity maturation by our vaccine may thus beof clinical significance.

In primary HIV-1 infection, neutralizing antibodies to autologous virususually develop slowly and peak after the decrease of viremia. (Koup RA,et al. J Virol 1994; 68: 4650-5). Promoting the induction of suchantibodies in primary infection or at any time during HIV-1 disease maydecelerate the virus induced pathology. It is of note that in all thevaccinees there was. an increase in serum neutralization of autologousand of primary isolates starting at 3 months and peaking at 36-172 foldincrease at 12 months (Table 37; FIGS. 14A-14G). So far no other vaccinehas induced such a potent neutralization of autologous and of primaryisolates in the HIV-1 log reduction assay or in the resting cell assay.(Zolla-Pazner S, et al. AIDS Res Hum Retrov 1995; 11: 1449-58). Theneutralization did, however, appear to be dependent on continuousboosters of the vaccine. In patient #7, for example, the neutralizationtiter dropped 9 months after the last vaccine boost.

Long term survivors have a lower virus load (Saag M S, Holodniy M, etal. Nat Med 1996; 625-29; Mellors J W, et al. Ann Intern Med 1995; 122:573-79; Mellors J W, et al. Science 1996; 272: 1167-70; Ho. D D, et al.Science 1996; 272: 1124-5) and several studies have shown a dosedependent effect of maternal plasma viremia on the rate of HIV-1transmission to the baby. (Weiser B, et al. Proc Natl Acad Sci USA 1994;91: 8037-41; Dickover K, et al. Cell Biochem 1995; S21B, 233). We haveshown a maintenance of an undetectable viral load or a decrease of viralloads in all vaccinees (FIGS. 15A-15G). In two patients with theshortest course of immunization a decrease of viral load to undetectablelevels was followed 6 months, respectively, 9 months after the lastvaccine boost by an increase in viral load. This renewed viremia wasdocumented to be associated with the loss of antibody affinity (FIGS.13A and 13B & 15A-15G).

In principal, subunit vaccines have been more effective in inducingantibody responses, while non HIV-1 live vaccine expressing HIV-1epitopes induced cytotoxic Tcells (CTLs) but did not elicit high titersof neutralizing antibodies. (Abimiku A G, et al. Nature Medicine 1995;1: 321-29; Perales M A, et al. J. AIDS & Human Retrov 1995; 10: 27-35.The breadth of the immune responses enhanced by our vaccine includinghigh affinity and neutralizing immunity to autologous and primaryisolates and the reduction in plasma viremia suggest that irrespectiveof the mechanism of action, this vaccine has a favorable influence onparameters associated with improved prognosis.

All publications mentioned hereinabove are hereby incorporated in theirentirety.

While the foregoing invention has been described in some detail forpurposed of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of the disclosure that various changesin form and detail can be made without departing from the true scope ofthe invention in the appended claims.

16 13 amino acid single linear <Unknown> peptide No 1 Lys Arg Ile HisIle Gly Pro Gly Arg Ala Phe Tyr Thr 1 5 10 9 amino acid single linear<Unknown> peptide No 2 Ile Thr Ile Gly Pro Gly Arg Ala Cys 1 5 9 aminoacid single linear <Unknown> peptide No 3 Ile Ala Ile Gly Pro Gly ArgAla Cys 1 5 9 amino acid single linear <Unknown> peptide No 4 Ile HisIle Gly Pro Gly Arg Ala Cys 1 5 19 amino acid single linear <Unknown>peptide No 5 Gly Pro Gly Arg Ala Phe Gly Pro Gly Arg 1 5 10 Ala Phe GlyPro Gly Arg Ala Phe Cys 15 13 amino acid single linear <Unknown> peptideNo 6 Arg Ser Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Ala 1 5 10 13 aminoacid single linear <Unknown> peptide No 7 Lys Ser Ile Thr Lys Gly ProGly Arg Val Ile Tyr Ala 1 5 10 13 amino acid single linear <Unknown>peptide No 8 Lys Gly Ile Ala Ile Gly Pro Gly Arg Thr Leu Tyr Ala 1 5 1013 amino acid single linear <Unknown> peptide No 9 Ser Arg Val Thr LeuGly Pro Gly Arg Val Trp Tyr Thr 1 5 10 7 amino acid single linear<Unknown> peptide No 10 Leu Leu Glu Asp Lys Trp Ala 1 5 6 amino acidsingle linear <Unknown> peptide No 11 Arg Pro Met Thr Tyr Lys 1 5 6amino acid single linear <Unknown> peptide No 12 Gly Gly Lys Trp Ser Lys1 5 7 amino acid single linear <Unknown> peptide No 13 Pro Gly Pro GlyIle Arg Tyr 1 5 8 amino acid single linear <Unknown> peptide No 14 GlyPro Gly Ile Gly Pro Gly Val 1 5 10 amino acid single linear <Unknown>peptide No 15 Ile His Ile Gly Pro Gly Arg Ala Phe Tyr 1 5 10 6 aminoacid single linear <Unknown> peptide No 16 Gly Pro Gly Arg Ala Phe 1 5

What is claimed is:
 1. A method for treating a mammal at risk for HIVinfection comprising administering to said mammal a peptide compositioncomprising peptides KRIHIGPGRAFYT (SEQ ID NO:1), RSIHIGPGRAFYA (SEQ IDNO:6), KSITKGPGRVIYA (SEQ ID NO:7), KGIAIGPGRTLYA (SEQ ID NO:8) andSRVTLGPGRVWYT (SEQ ID NO:9), wherein each peptide is coupled to a PPDcarrier, and wherein the peptide composition is administered to saidmammal in an amount effective to reduce the level of HIV titers in saidmammal upon subsequent HIV infection.
 2. The method of claim 1, whereinthe composition is administered intradermally.
 3. The method of claim 1,wherein a PPD carrier is conjugated to each peptide via glutalderhyde.